Methods and compositions for the treatment of angiogenic diseases

ABSTRACT

Provided are agents that can bind to copper, and form a tripartite complex with protein, and the use of these agents in the prevention and treatment of diseases with a vascular component, such as solid tumors. Compositions and methods for combination therapy of these diseases, including cancer, as well as therapeutic kits, are also provided.

The present application is a continuation of U.S. patent applicationSer. No. 09/389,435, filed Sep. 3, 1999, now U.S. Pat. No. 6,703,050which claims the priority date of provisional application Ser. No.60/099,103, filed Sep. 4, 1998, and provisional application Ser. No.60/101,759, filed Sep. 25, 1998, the entire disclosures of which areincorporated herein by reference without disclaimer.

The government owns rights in the present invention pursuant to grantnumber FD-U-000505 from the Food and Drug Administration, and grantnumber RO3-CA77122-01 from the National Institutes of Health.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of diseaseprophylaxis and therapy. More particularly, it concerns agents that canbind to copper, in certain aspects forming a tripartite complex withprotein, and the use of these agents in the prevention and treatment ofdiseases with a vascular component, such as cancer. Compositions andmethods for combination therapy of these diseases, as well astherapeutic kits, are also provided.

2. Description of Related Art

Solid tumors require blood vessel proliferation (angiogenesis) forsustained growth in order to maintain adequate nutrition to other thanthe most peripheral cell layers (Hayes, 1994; Horak et al., 1993;Parangi et al., 1996). Normal adult human tissues, on the other hand,require little new blood vessel growth, except for wound repair,regeneration following trauma or surgery, and proliferation of the innerlining of the uterus during the menstrual cycle. Thus, dependency onangiogenesis is a fundamental difference between tumor and normaltissue. This difference is quantitatively more striking than thedifferences in cell replication and cell death rates, on which manycytoreductive chemotherapies depend. As a result of tumor dependency onangiogenesis, the concept of anti-angiogenic therapy for malignancieswas developed (Folkman, 1995a; Folkman, 1995b; Hanahan and Folkman,1996).

Copper is both a requirement and a potent stimulus for angiogenesis, asshown by studies of neovascularization in the rabbit cornea (Parke etal., 1988). During prostaglandin E1 (PGE1)-induced angiogenesis in therabbit cornea, copper accumulates at the site where angiogenesis occurs(Parke et al., 1988). Conversely, in copper deficient rabbits,angiogenesis in the rabbit cornea in response to PGE1 is greatlyreduced. In the rabbit cornea, copper for angiogenesis can be suppliedby ceruloplasmin (a copper protein) as well as by dissolved coppersulfate, while apoceruloplasmin (ceruloplasmin without copper) does notsupport angiogenesis (Gullino, 1986). Additional studies have also shownthat copper is an important angiogenic agent (Raju et al., 1982; Zicheet al., 1982). These studies all support the concept that unbound copperis required for angiogenesis.

Several years ago, some animal tumor model studies were carried outusing an anti-copper approach (Brem et al., 1990a; 1990b; Yoshida etal., 1995). The chelator penicillamine plus a low-copper diet were usedto lower copper levels in rats and rabbits with implanted intracerebraltumors. However, the animals treated with the low-copper regimen, whileshowing reduction in tumor size, did not show improved survival overuntreated controls.

Penicillamine therapy has also been reported to be associated withsignificant side effects, including nausea and abdominal discomfort, andmore serious side effects such as leukopenia and thrombocytopenia, whichcan lead to aplastic anemia. Nephrotic syndrome has also been reportedin certain instances.

There are numerous other examples of diseases characterized by aberrantangiogenesis. One example of such a disease mediated by angiogenesis isocular neovascular disease. This disease is characterized by invasion ofnew blood vessels into the structures of the eye such as the retina orcornea. It is the most common cause of blindness and is involved inapproximately twenty eye diseases. In age-related macular degeneration,the associated visual problems are caused by an ingrowth of chorioidalcapillaries through defects in Bruch's membrane with proliferation offibrovascular tissue beneath the retinal pigment epithelium.

Another disease in which angiogenesis is believed to be involved isrheumatoid arthritis. The blood vessels in the synovial lining of thejoints undergo angiogenesis. In addition to forming new vascularnetworks, the endothelial cells release factors and reactive oxygenspecies that lead to pannus growth and cartilage destruction. Thefactors involved in angiogenesis may actively contribute to, and helpmaintain, the chronically inflamed state of rheumatoid arthritis.

There remains a need in the art for agents that effectively prevent,slow the onset or progression of, reduce or treat diseases characterizedby angiogenesis, such as cancer, macular degeneration, and rheumatoidarthritis. The development of successful prophylactic or therapeuticagents that reduce the level of copper in vivo, without the side effectsand risks associated with other copper reducing agents, would representa particularly significant advance.

SUMMARY OF THE INVENTION

The present invention overcomes these and other deficiencies present inthe art by providing methods and compositions that, in certain aspectsof the invention, slow the onset or progression of or even preventdiseases characterized by aberrant vascularization or angiogenesis, suchas cancer, and in other aspects of the invention, reduce or treat suchdiseases. This is accomplished by the provision of agents that reducethe level of copper in vivo, in preferred aspects through the formationof a tripartite agent-copper-protein complex. The compositions andmethods of the present invention accomplish this without the sideeffects and risks associated with other copper reducing agents.

As used herein, the term “aberrant vascularization” or “aberrantangiogenesis” will be understood to include abnormal neovascularization,including the formation of new blood vessels, larger blood vessels, morebranched blood vessels (intussusception), and any and all mechanismsthat lead to inappropriate or increased blood carrying capacity to adiseased tissue or site. The agents of the present invention will beunderstood to counteract “aberrant vascularization” or “aberrantangiogenesis”, irrespective of the actual mechanism of action.

The present invention provides a method of delaying the onset of orpreventing cancer in an animal, comprising administering to an animal atrisk for developing cancer a biologically effective amount of at least afirst pharmaceutical composition comprising at least a firstprophylactic agent that binds copper and forms an agent-copper-proteincomplex upon administration to the animal. The protein in theagent-copper-protein complex can be a food protein, or a serum protein,such as serum albumin. The present invention also provides a method ofdelaying the onset of or preventing cancer in a human subject,comprising administering to a human subject at risk for developingcancer a biologically effective amount of at least a firstpharmaceutical composition comprising at least a first prophylacticagent that binds copper and forms an agent-copper-protein complex uponadministration to the human subject.

The present invention also provides a method of delaying the onset of orpreventing cancer in an animal or human subject, comprising selecting ananimal or human subject at risk for developing cancer, and administeringto the animal or human subject at risk for developing cancer atherapeutically effective amount of at least a first pharmaceuticalcomposition comprising at least a first prophylactic agent that bindscopper and forms an agent-copper-protein complex upon administration tothe animal or human subject. The “selecting” step may be accomplished,e.g., by identifying relatives of cancer patients and/or identifyingsusceptible subjects by genetic testing.

In certain aspects of the invention, the at least a first agent is athiomolybdate compound. Preferred thiomolybdate compounds for use in thepresent invention include, but are not limited to,dodecathiodimolybdate, tetrathiomolybdate, iron octathiodimolybdate,trithiomolybdate, dithiomolybdate or monothiomolybdate. In particularlypreferred aspects of the invention, the at least a first agent istetrathiomolybdate. In other embodiments of the invention, thethiomolybdate compound comprises at least a first iron atom and/or atleast a first oxygen atom. It will be understood that complete oxidationof the thiomolybdate compound should preferably be avoided.

In other aspects of the invention, the thiomolybdate compound isassociated with at least a first agent effective to form a stabilizedthiomolybdate compound. Exemplary stabilizing agents are carbohydratemolecules, such as a monosaccharide, a disaccharide, a trisaccharide, anoligosaccharide or a polysaccharide. In preferred embodiments, thethiomolybdate compound is associated with at least a first sucrosemolecule, such as forming a thiomolybdate compound associated withbetween about 5 and about 200 sucrose molecules, and in other preferredembodiments of the invention, the thiomolybdate compound is associatedwith about 30 sucrose molecules.

Thus, the present invention provides a method of delaying the onset ofor preventing cancer in an animal, or in a human subject, comprisingadministering to the animal or subject a biologically effective amountof at least a first pharmaceutical composition comprisingdodecathiodimolybdate, tetrathiomolybdate, iron octathiodimolybdate,trithiomolybdate, dithiomolybdate, monothiomolybdate or acarbohydrate-associated complex thereof. The invention also provides amethod of delaying the onset of or preventing cancer in an animal, or ina human subject, comprising administering to the animal or subject abiologically effective amount of a pharmaceutical composition comprisingtetrathiomolybdate.

The agents for use in the present invention, such as tetrathiomolybdate,lower copper levels by forming a “tripartite agent-copper-proteincomplex” that is subsequently cleared from the body. The copper bound inthese “tripartite agent-copper-protein complexes” is not reversiblyreleased from these complexes, and are thus distinguished fromreversible bipartite copper chelation.

As used herein, the term “biologically effective amount” will beunderstood to mean an amount of an agent that binds copper and forms anagent-copper-protein complex effective to delay the onset of or preventdiseases associated with aberrant vascularization, preferably cancer,upon administration to selected animals or patients. Thus in thepreventative aspects of the present invention, the term“prophylactically effective amount” can also be used to describe theamount of the instant compositions effective to delay the onset of orprevent diseases and/or cancer upon administration to selected animalsor patients.

Alternatively, in the therapeutic aspects of the present invention, theterm “therapeutically effective amount” can also be used to describe theamount of the instant compositions effective to delay the onset of orprevent cancer. In terms of cancer treatment, amounts are effective toslow the growth of a tumor; to stop the growth of a tumor; tospecifically induce necrosis in at least a portion of a tumor; and/or toinduce tumor regression or remission upon administration to selectedanimals or patients. Such effects are achieved while exhibitingnegligible or manageable adverse side effects on normal, healthy tissuesof the animal or patient. Thus, the “therapeutically effective amount”can vary from animal to animal or patient to patient, depending on anumber of factors including, but not limited to, the extent of diseaseand the size of patient. All such dosing issues can be routinelyaddressed by the attending physician in light of the present disclosure.

The present invention further provides a method of treating an animalhaving at least a first tumor, comprising administering to the animal abiologically effective amount of at least a first pharmaceuticalcomposition comprising at least a first agent that binds copper andforms an agent-copper-protein complex upon administration to the animal.Additionally, the present invention provides a method of treating ahuman patient having at least a first tumor, comprising administering tothe patient a therapeutically effective amount of at least a firstpharmaceutical composition comprising at least a first therapeutic agentthat binds copper and forms an agent-copper-protein complex uponadministration to the patient. In certain aspects of the invention, theat least a first agent is a thiomolybdate compound, such asdodecathiodimolybdate, tetrathiomolybdate, iron octathiodimolybdate,trithiomolybdate, dithiomolybdate, monothiomolybdate or acarbohydrate-associated complex thereof. In particularly preferredembodiments of the invention, the at least a first agent istetrathiomolybdate. Thus, the present invention provides a method oftreating an animal or a human patient having at least a first tumor,comprising administering to the patient a therapeutically effectiveamount of dodecathiodimolybdate, tetrathiomolybdate, ironoctathiodimolybdate, trithiomolybdate, dithiomolybdate ormonothiomolybdate. The present invention also provides a method oftreating an animal or a human patient having at least a first tumor,comprising administering to the patient a therapeutically effectiveamount of tetrathiomolybdate. In various therapeutic embodiments of thepresent invention, the preferred at least a first agent is thustetrathiomolybdate.

Thus, the present invention provides a method of prophylactic ortherapeutic intervention in a human subject having or at risk fordeveloping a tumor, comprising administering to the subject aprophylactically or therapeutically effective amount of at least a firstagent that binds copper and forms an agent-copper-protein complex. Thepresent invention further provides a method of prophylactic ortherapeutic intervention in a human subject having or at risk fordeveloping a tumor, comprising administering to the subject aprophylactically or therapeutically effective amount ofdodecathiodimolybdate, tetrathiomolybdate, iron octathiodimolybdate,trithiomolybdate, dithiomolybdate or monothiomolybdate. The presentinvention additionally provides a method of prophylactic or therapeuticintervention in a human subject having or at risk for developing atumor, comprising administering to the subject a prophylactically ortherapeutically effective amount of tetrathiomolybdate. The presentinvention also provides a method of treating a human patient having atumor, preferably a vascularized tumor, comprising selecting a patientthat would benefit from copper-protein complexation, and administering atherapeutically effective amount of at least a first copper-complexingagent effective to form agent-copper-protein complexes in the patient.

As used throughout the entire application, the terms “a” and “an” areused in the sense that they mean “at least one”, “at least a first”,“one or more” or “a plurality” of the referenced components or steps,except in instances wherein an upper limit is thereafter specificallystated. Therefore “an agent that binds copper and forms anagent-copper-protein complex” means “at least a first agent that bindscopper and forms an agent-copper-protein complex”. The operable limitsand parameters of combinations, as with the amounts of any single agent,will be known to those of ordinary skill in the art in light of thepresent disclosure.

In certain aspects of the invention, the biologically effective amountof the at least a first agent is between about 20 mg and about 200 mgper patient. In certain aspects of the invention, the therapeuticallyeffective amount of the at least a first agent is between about 20 mgand about 200 mg per patient over a therapeutically effective time orperiod. In general, the agent is administered to the patient daily, andthus in these embodiments of the invention, the biologically ortherapeutically effective amount of the at least a first agent isbetween about 20 mg and about 200 mg per patient per day. “Between about20 mg and about 200 mg” includes all values in this range, and thusincludes amounts of about 25 mg, about 30 mg, about 40 mg, about 50 mg,about 60 mg, about 70 mg, about 75 mg, about 80 mg, about 90 mg, about100 mg, about 100 mg, about 120 mg, about 125 mg, about 130 mg, about140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about190 mg or about 195 mg. “About” will be understood to include valuesabove and below the given number. Thus, “about 20 mg” will be understoodto include about 18 mg, about 19 mg, about 21 mg and about 22 mg or so,and “about 200 mg” will be understood to include about 201 mg, about 202mg, about 203 mg and about 204 mg or so.

In other embodiments of the invention, the biologically ortherapeutically effective amount of the at least a first agent isbetween about 20 mg and about 190 mg, between about 20 mg and about 180mg, between about 20 mg and about 170 mg, between about 20 mg and about160 mg, between about 20 mg and about 150 mg, between about 20 mg andabout 140 mg, between about 20 mg and about 130 mg, between about 20 mgand about 120 mg, between about 20 mg and about 110 mg, between about 20mg and about 100 mg, between about 20 mg and about 90 mg, between about20 mg and about 80 mg, between about 20 mg and about 70 mg, betweenabout 20 mg and about 60 mg, between about 20 mg and about 50 mg,between about 20 mg and about 40 mg, between about 20 mg and about 30mg, between about 30 mg and about 200 mg, between about 40 mg and about200 mg, between about 50 mg and about 200 mg, between about 60 mg andabout 200 mg, between about 70 mg and about 200 mg, between about 80 mgand about 200 mg, between about 90 mg and about 200 mg, between about100 mg and about 200 mg, between about 110 mg and about 200 mg, betweenabout 120 mg and about 200 mg, between about 130 mg and about 200 mg,between about 140 mg and about 200 mg, between about 150 mg and about200 mg, between about 160 mg and about 200 mg, between about 170 mg andabout 200 mg, between about 180 mg and about 200 mg, between about 190mg and about 200 mg, between about 30 mg and about 190 mg, between about40 mg and about 180 mg, between about 50 mg and about 170 mg, betweenabout 60 mg and about 160 mg, between about 70 mg and about 150 mg,between about 80 mg and about 140 mg, between about 90 mg and about 130mg, between about 100 mg and about 120 mg, between about 125 mg andabout 200 mg or between about 150 mg and about 180 mg per patient perday.

These values can also be expressed in terms of mg/kg of body weight. Asdescribed above, the biologically or therapeutically effective amountcan vary depending on the size of the animal or human patient. However,taking the average weight of a human male as about 70 kg, thebiologically or therapeutically effective amount of the agent that bindscopper and forms an agent-copper-protein complex for an average humanmale would be between about 0.3 mg/kg and about 3 mg/kg or so.

The compositions and methods of the present invention can be used totreat all forms of solid tumors having a vascular component, inparticular those that are extensively vascularized, although this is notrequired in all aspects of the invention. Solid tumors, such ascarcinomas and sarcomas, are exemplary of the types of tumors amenableto treatment with the instant compositions and methods. Particularexamples of the types of tumors that may be prevented or treated usingthe present invention include, but are not limited to, renal, lung,breast, colon, prostate, stomach, liver, pancreas, esophagus, brain andlarynx tumors, as well as angiosarcomas and chondrosarcomas.

Tumors of various sizes may also be treated using the present invention.Thus, small tumors, including metastatic tumors, exemplified by tumorsthat are about 1 cm in diameter or less, medium or moderate tumors,exemplified by tumors that are between about 1 cm and about 5 cm indiameter, and large tumors, exemplified by tumors that are about 5 cm indiameter or greater are contemplated for treatment using the presentinvention. In the context of the present invention, the term “avascularized tumor” most preferably means a vascularized, malignanttumor, solid tumor or “cancer”.

Since, as discussed above, the instant compositions and methods are notcompletely specific to any particular tumor type, patients having morethan one type of tumor may also be treated by the present invention.Thus, patients having at least a first and at least a second distincttype of tumor, exemplified by, but not limited to, a breast tumor and achondrosarcoma or a renal tumor and a lung tumor, at least threedistinct types of tumors, at least four distinct types of tumors, atleast five distinct types of tumors or more are contemplated fortreatment using the present invention.

In certain preferred aspects of the present invention, the at least afirst agent is administered to the patient orally. However, other routesof administration are contemplated, including, but not limited to,intravenous, intramuscular and subcutaneous injections, slow releaseformulations and the like.

In certain embodiments of the invention, the methods further compriseadministering to the human subject a therapeutically effective amount ofat least a second anti-cancer agent. Exemplary of the additionalanti-cancer agents contemplated for use are chemotherapeutic agents,radiotherapeutic agents, distinct copper chelating agents,anti-angiogenic agents, apoptosis-inducing agents or zinc compounds. Infurther aspects of the present invention, the methods further comprisesubjecting the patient to surgery or radiotherapy.

The at least a first anti-cancer agent may be a “chemotherapeuticagent”. As used herein, the term “chemotherapeutic agent” is used torefer to a classical chemotherapeutic agent or drug used in thetreatment of malignancies. This term is used for simplicitynotwithstanding the fact that other compounds, including immunotoxins,may be technically described as a chemotherapeutic agent in that theyexert an anti-cancer effect. However, “chemotherapeutic” has come tohave a distinct meaning in the art and is being used according to thisstandard meaning. “Chemotherapeutics” in the context of the presentapplication therefore do not generally refer to immunotoxins,radiotherapeutic agents and such like, despite their operationaloverlap.

A number of exemplary chemotherapeutic agents are listed in Table 1.Those of ordinary skill in the art will readily understand the uses andappropriate doses of chemotherapeutic agents, although the doses maywell be reduced when used in combination with the present invention. Anew class of drugs that may also be termed “chemotherapeutic agents” areagents that induce apoptosis. Any one or more of such drugs, includinggenes, vectors and antisense constructs, as appropriate, may also beused in conjunction with the present invention.

In certain aspects of the present invention, the methods compriseadministering a therapeutically effective amount of a zinc compound tothe animal or human subject. In preferred embodiments, the methodscomprise administering the at least a first agent to the animal or humansubject in an amount and for a time effective to reduce the level ofcopper in the animal or human subject to about 20% of the level ofcopper in the animal or human subject prior to administration of the atleast a first agent, and administering to the animal or human subject atherapeutically effective amount of a zinc compound, such as zincacetate. In yet other preferred aspects, the therapeutically effectiveamount of a zinc compound is administered to the animal or human subjectfor a period of time effective to maintain the level of copper in theanimal or human subject at about 20% of the level of copper in theanimal or human subject prior to administration of the at least a firstagent.

The present invention also provides methods of treating cancer in ahuman subject, comprising administering a pharmaceutical compositioncomprising tetrathiomolybdate to a human subject having cancer in anamount and for a time effective to reduce the level of copper in thehuman subject to about 20% of the level of copper in the human subjectprior to administration of tetrathiomolybdate, and administering to thehuman subject a pharmaceutical composition comprising a therapeuticallyeffective amount of a zinc compound.

The present invention also provides methods of treating or preventing adisease characterized by aberrant vascularization in an animal or ahuman patient, comprising administering to an animal or a human patienthaving or at risk for developing a disease characterized by aberrantvascularization a therapeutically effective amount of at least a firstpharmaceutical composition comprising at least a first agent that bindscopper and forms an agent-copper-protein complex. In preferredembodiments of the invention, the disease is cancer, wet type maculardegeneration or rheumatoid arthritis.

Further provided by the present invention are methods of treating orpreventing wet type macular degeneration in an animal or human patient,comprising administering to an animal or human patient having or at riskfor developing wet type macular degeneration a therapeutically effectiveamount of at least a first pharmaceutical composition comprising atleast a first agent that binds copper and forms an agent-copper-proteincomplex. In certain aspects of the invention, the at least a first agentis a thiomolybdate compound, such as dodecathiodimolybdate,tetrathiomolybdate, iron octathiodimolybdate, trithiomolybdate,dithiomolybdate, monothiomolybdate, or a carbohydrate-associated complexof any of the foregoing.

Additionally, the present invention provides methods of treating orpreventing rheumatoid arthritis in an animal or human patient,comprising administering to an animal or human patient having or at riskfor developing rheumatoid arthritis a therapeutically effective amountof at least a first pharmaceutical composition comprising at least afirst agent that binds copper and forms an agent-copper-protein complex.In preferred aspects of the invention, the at least a first agent is athiomolybdate compound, such as dodecathiodimolybdate,tetrathiomolybdate, iron octathiodimolybdate, trithiomolybdate,dithiomolybdate, monothiomolybdate, or a carbohydrate-associated complexof any of the foregoing.

The present invention also provides a composition, or a pharmaceuticalcomposition, comprising a combined therapeutic amount of at least afirst agent that binds copper and forms an agent-copper-protein complexand at least a second anti-cancer agent. In certain aspects of theinvention, the at least a first agent is a thiomolybdate compound, suchas dodecathiodimolybdate, tetrathiomolybdate, iron octathiodimolybdate,trithiomolybdate, dithiomolybdate, monothiomolybdate, or acarbohydrate-associated complex thereof. In preferred aspects of theinvention, the at least a first agent that binds copper and forms anagent-copper-protein complex is tetrathiomolybdate. In further preferredembodiments, the at least a second anti-cancer agent is achemotherapeutic agent, a radiotherapeutic agent, a distinct copperchelating agent, an anti-angiogenic agent, an apoptosis-inducing agentor a zinc compound. Thus, the present invention also provides acomposition comprising a combined therapeutic amount ofdodecathiodimolybdate, tetrathiomolybdate, iron octathiodimolybdate,trithiomolybdate, dithiomolybdate or monothiomolybdate and at least asecond anti-cancer agent. The present invention additionally provides acomposition comprising a combined therapeutic amount oftetrathiomolybdate and at least a second anti-cancer agent.Pharmaceutical compositions comprising a thiomolybdate compoundassociated with at least a first carbohydrate molecule are alsoprovided.

Also provided is a therapeutic kit comprising, in at least a firstsuitable container, a therapeutically effective combined amount of atleast a first agent that binds copper and forms an agent-copper-proteincomplex, and at least a second anti-cancer agent. In certain aspects ofthe invention, the at least a first agent is a thiomolybdate compound,such as dodecathiodimolybdate, tetrathiomolybdate, ironoctathiodimolybdate, trithiomolybdate, dithiomolybdate ormonothiomolybdate. In a preferred embodiment of the therapeutic kits ofthe present invention, the at least a first agent that binds copper andforms an agent-copper-protein complex is tetrathiomolybdate. In otheraspects of the invention, the thiomolybdate compound is associated withat least a first carbohydrate molecule. In additional aspects of thetherapeutic kits of the invention, the at least a second anti-canceragent is a chemotherapeutic agent, a radiotherapeutic agent, a distinctcopper chelating agent, an anti-angiogenic agent, an apoptosis-inducingagent or a zinc compound. In particular embodiments of the presentinvention, the at least a first agent that binds copper and forms anagent-copper-protein complex and the at least a second anti-cancer agentare comprised in at least a first and at least a second separatecontainers.

The present invention also provides therapeutic kits comprising, in atleast a first suitable container, a therapeutically effective combinedamount of dodecathiodimolybdate, tetrathiomolybdate, ironoctathiodimolybdate, trithiomolybdate, dithiomolybdate,monothiomolybdate, or a carbohydrate-associated complex thereof, and atleast a second anti-cancer agent. Further provided are therapeutic kitscomprising, in at least a first suitable container, a therapeuticallyeffective combined amount of tetrathiomolybdate and at least a secondanti-cancer agent.

The present invention additionally provides compositions comprising athiomolybdate compound associated with at least a first stabilizingmolecule, such as a carbohydrate molecule, or a“thiomolybdate-carbohydrate complex”. In certain aspects of theinvention, the thiomolybdate compound comprises at least a first ironresidue and/or at least a first oxygen residue. In other aspects, theratio of carbohydrate molecules, or sugar units, to the thiomolybdatecompound is between about 200 to 1 and about 5 to 1. It will beunderstood that all ratios within this range are also included, such asratios of about 190 to 1, about 180 to 1, about 175 to 1, about 170 to1, about 160 to 1, about 150 to 1, about 140 to 1, about 130 to 1, about125 to 1, about 120 to 1, about 110 to 1, about 100 to 1, about 90 to 1,about 80 to 1, about 75 to 1, about 70 to 1, about 60 to 1, about 50 to1, about 40 to 1, about 30 to 1, about 25 to 1, about 20 to 1, about 15to 1 and about 10 to 1. In preferred aspects of the invention, the ratioof carbohydrate molecules to the thiomolybdate compound is about 30 to1.

In further embodiments of the present invention, the at least a firstcarbohydrate molecule is a monosaccharide. In other aspects, the atleast a first carbohydrate molecule is a disaccharide, such as sucrose.In yet other aspects, the at least a first carbohydrate molecule is anoligosaccharide. In certain embodiments, the thiomolybdate compound isassociated with at least a first and at least a second distinctcarbohydrate molecule.

The thiomolybdate compound may be non-covalently bonded to the at leasta first stabilizing agent, e.g., carbohydrate molecule, such as byhydrogen bonding. In other aspects of the invention, the thiomolybdatecompound is covalently bonded to at least a first stabilizing agent,e.g., carbohydrate molecule. In still other aspects, the thiomolybdatecompound is complexed with the at least a first carbohydrate molecule.In preferred aspects, the thiomolybdate compound isdodecathiodimolybdate, tetrathiomolybdate, iron octathiodimolybdate,trithiomolybdate, dithiomolybdate or monothiomolybdate. In particularlypreferred aspects, the thiomolybdate compound is tetrathiomolybdate. Infurther aspects, the compositions comprises a zinc compound. In otherpreferred aspects, the composition is dispersed in a pharmaceuticallyacceptable excipient. The present invention thus provides stabilizedtetrathiomolybdate compositions comprising tetrathiomolybdate associatedwith about 30 sucrose molecules.

The agents that bind copper and form an agent-copper-protein complex, asdescribed herein, can be used in treating or preventing a diseasecharacterized by aberrant vascularization, including, but not limitedto, cancer, wet type macular degeneration or rheumatoid arthritis. Useof the agents that binds copper and forms an agent-copper-proteincomplex in the manufacture of medicaments for treating or preventing adisease characterized by aberrant vascularization, including, but notlimited to, cancer, wet type macular degeneration or rheumatoidarthritis, are thus also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. Disease-free survival of TM-treated and control animals as afunction of age. Daily TM treatments begun for each animal atapproximately 100 days of age. Treatment (□) and control (Δ) groups aregenetically identical cancer prone HER2-neu transgenic mice. Percentageof animals that are disease free is shown on the vertical axis, age indays is shown on the horizontal axis.

FIG. 2. Ceruloplasmin activity as a function of time in TM-treated mice.A spectrophotometric assay of activity was used for the serum oftreatment and control mice at regular intervals during treatment.Fraction of control ceruloplasmin is shown on the vertical axis, days ofTM therapy is shown on the horizontal axis.

FIG. 3. Tumor size over time in nude mice injected with SUM149 breastcancer cell line cells. Three groups of five mice were injected with 10⁶SUM149 breast cancer cells, and two of the groups were given 1.2mg/day/mouse of TM via drinking water, starting at day −7. After 34days, the TM was skipped in these two groups of mice, and then restartedat half of the previous dose (0.6 mg/day/mouse; Δ) or the full dose (1.2mg/day/mouse; ◯). Control mice (⋄) received no TM. The mean tumorcross-sectional area (mm²) is shown on the vertical axis, and time(days) is shown on the horizontal axis.

FIG. 4A, FIG. 4B and FIG. 4C. Ceruloplasmin as a surrogate marker oftotal-body copper status. Rates of decrease of the ratio of Cp at time tto baseline Cp, as a function of days on TM therapy, are depicted fordose levels I (FIG. 4A), II (FIG. 4B), and III (FIG. 4C). The averagetime to 50% reduction of Cp is 30 days.

FIG. 5A and FIG. 5B. Management of long-term therapy with TM to maintaina Cp target of 20% of baseline in two patients. FIG. 5A. Patientrequired decrease in dose at 2 time points 60 days apart. In order toprevent the Cp from falling below 5 mg/d, this patient will likelyrequire a decrease in TM dose in the future. FIG. 5B. TM dose wasincreased after day 100 to prevent a drifting of the Cp above targetrange. Heterogeneity of diet and tumor behavior (such as tumor celllysis) may account for the individual variability in dose adjustmentneeds.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Solid tumors account for more than 90% of all cancers in man (Shockleyet al., 1991). The therapeutic uses of monoclonal antibodies andimmunotoxins have been investigated in the therapy of lymphomas andleukemias (Lowder et al., 1987; Vitetta et al., 1991), but have beendisappointingly ineffective in clinical trials against solid tumors(Byers and Baldwin, 1988; Abrams and Oldham, 1985).

A principal reason for the ineffectiveness of antibody-based treatmentsis that macromolecules are not readily transported into solid tumors(Sands, 1988; Epenetos et al., 1986). Even when these molecules get intothe tumor mass, they fail to distribute evenly due to the presence oftight junctions between tumor cells (Dvorak et al., 1991), fibrousstroma (Baxter et al., 1991), interstitial pressure gradients (Jain,1990) and binding site barriers (Juweid et al., 1992).

All too often in cancer control, a high-functioning individual is copingwith the high likelihood of a clinical cancer diagnosis or theinexorable progression of largely asymptomatic cancer to a lethal stage.At the cellular level, in spite of the diversity of the clinicalsettings, the incipient and existing tumors will all require new bloodvessel growth or angiogenesis to affect the quality of life of theirhosts. Therefore, the development of successful anti-angiogenictherapies may have the effect of preventing an emerging tumor frombecoming clinically relevant, or may allow a prolonged asymptomaticstate with stable metastatic disease. In addition, such therapy may alsohave the effect of down-staging overt tumors.

It has been known since the 1970's that copper is a co-factor inangiogenesis. Many key angiogenic mediators such as FGF, angiogenin, andSPARC bind or interact with copper in their pro-angiogenic state. Theconcept of anti-angiogenic treatment for solid tumors (Folkman, 1972;1995c; 1997), has a firm rationale and shows efficacy in animal tumormodels (Volpert et al., 1996; Millauer et al., 1996; Warren et al.,1995; Borgstom et al., 1996; 1998; Yuan et al., 1996; O'Reilly et al.,1997; Benjamin et al., 1999 and Merajver et al., 1998). Compounds thatinterfere with critical steps in the angiogenesis cascade are reachingthe clinic (Marshall et al., 1997). The steps required for successfultumor angiogenesis at the primary and metastatic sites are diverse, andthey depend on an imbalance between angiogenesis activators(Iruela-Arispe and Dvorak, 1997; Hanahan and Folkman, 1996) such as VEGFand bFGF, and inhibitors such as thrombospondin-1 (TSP-1) (Volpert etal., 1995; Salnikow et al., 1997; Guo et al., 1997; Schapira andSchapira, 1983; Qian et al., 1997), angiostatin, (O'Reilly et al., 1994;Lannutti et al., 1997; Sim et al., 1997) and endostatin (O'Reilly etal., 1997). The relative importance of the different angiogenesismodulating molecules in different tissues may determine the relativepotency of anti-angiogenic compounds to elicit a response both at theprimary and metastatic sites.

It has been amply demonstrated that copper is required for angiogenesis(Parke et al., 1988; Raju et al., 1982; Ziche et al., 1982). Brem et al.(1990a, b), and more recently Yoshida et al. (1995) attempted to use ananti-copper approach in the treatment of tumors in animal studies. Theyhave used a copper deficient diet combined with penicillamine therapy,called CDPT. The studies showed relative inhibition of growth of rabbitVX2 carcinoma in the rabbit brain (Brem et al., 1990b) and rat 9Lgliosarcoma in the rat brain (Brem et al., 1990a, Yoshida et al., 1995)as a result of such treatment, but, significantly, reported noimprovement in overall survival. The animals in Yoshida et al. weresacrificed before overall survival could be assessed. In the Brem et al.studies, death was due to accompanying intracerebral edema, which wassevere enough in treated animals to cause death at the same rate as theuntreated tumor did in control animals. Furthermore, in contrast to therabbit brain model, CDPT failed to inhibit tumor growth andvascularization of the VX2 carcinoma in the thigh muscle and inmetastases to lung. The lack of improvement in survival in brain tumorimplant models and the lack of effect in non-brain tumors seems to havediscouraged further work in this area.

The inventors reasoned that it would be very desirable to develop ananti-angiogenic strategy that would affect multiple activators ofangiogenesis, in order for it to be generally applicable to humantumors. Many anti-angiogenic proposals are directed against a singletarget. Since copper is a required co-factor for the function of manykey mediators of angiogenesis, such as bFGF (Watanabe et al., 1990;Engleka and Maciag, 1994; Shing, 1988; Patstone and Maher, 1996), VEGF,and angiogenin (Badet et al., 1989), the inventors developed ananti-angiogenic strategy for the treatment of cancer and other diseasescharacterized by aberrant angiogenesis based on the modulation oftotal-body copper status. The underlying basis of this work is that awindow of copper deficiency exists in which angiogenesis is impaired,but other copper dependent cellular processes are not affected enough tocause clinical toxicity. The inventors have succeeded in their clinicalobjectives, despite the documented failures of others using animalmodels.

The development of safe and effective prophylactic or therapeutic agentsthat reduce the level of copper in vivo has been an ongoing problem inthe art. The inventors hypothesis of an anti-copper, anti-angiogenicapproach to cancer therapy is that the level of copper required forangiogenesis is higher relative to that required for essential copperdependent cellular functions, such as heme synthesis, cytochromefunction, and incorporation of copper into enzymes and other proteins.The inventors reasoned that the unique and favorable characteristics oftetrathiomolybdate (TM) as an anti-copper agent, compared to otheranti-copper drugs, could make TM a non-toxic, efficacious new weapon inan anti-copper, anti-angiogenic therapy.

As described in detail below, for the past 20 years the inventors havedeveloped new anti-copper therapies for Wilson's disease, an autosomalrecessive disease of copper transport that results in abnormal copperaccumulation and toxicity. One of the drugs currently being used is TM,which shows unique and desirable properties of fast action,copper-specificity, and low toxicity (Brewer et al., 1991a; 1994b;1996), as well as a unique mechanism of action. TM forms a stabletripartite complex with copper and protein. If given with food, itcomplexes food copper with food protein and prevents absorption ofcopper from the gastrointestinal tract. There is endogenous secretion ofcopper in saliva and gastric secretions associated with food intake, andthis copper is also complexed by TM when it is taken with meals, therebypreventing copper re-absorption. Thus, patients are placed in a negativecopper balance immediately when TM is given with food. If TM is givenbetween meals, it is absorbed into the blood stream, where it complexeseither free or loosely bound copper with serum albumin. This TM-boundcopper fraction is no longer available for cellular uptake and has noknown biological activity.

The studies of the present invention indeed show TM to be a safe andeffective anti-cancer agent. TM showed efficacy in impairing thedevelopment of de novo mammary tumors in Her2-neu transgenic mice(Example 2), and showed no clinically overt toxicity as copper levelswere decreased to 10% of baseline. The present disclosure also detailsthe first human trial of an anti-copper approach to anti-angiogenesistherapy based on the use of tetrathiomolybdate in patients withmetastatic cancer (Examples 3 and 4). The phase I trial of TM, yieldinginformation on dose, dose response, evaluation of copper status inpatients, toxicity and efficacy, is surprisingly beneficial,particularly in light of the disappointing animal studies conducted byothers (Brem et al., 1990a, b; Yoshida et al., 1995). Novel approachesto following disease status in trials of anti-angiogenric compounds arealso disclosed.

I. Agents that Form a Tripartite Complex with Copper and Protein

A. Tetrathiomolybdate

1. Characterization and Mechanism of Action

Tetrathiomolybdate (TM) is a compound made up of molybdenum atomsurrounded by four sulfido groups. Discovery of the biological effectsof TM began with observations on cattle and sheep, in which theydeveloped copper deficiency when grazing on pasturages with highmolybdenum (Mo) content (Ferguson et al., 1943; Dick and Bull, 1945;Miller and Engel, 1960). It was established that administration ofsupplementary Mo impaired copper metabolism in ruminants (MacileseAmmerman et al., 1969); however, Mo had little effect on non-ruminantanimals such as rats (Mills et al., 1958; Cox et al., 1960). The answerto this puzzle came from observations which suggested that the Mo wasconverted to thiomolybdates in the rumen as a result of the high sulfidemetabolism there, and that thiomolybdates were the active anti-copperagents (Dick et al., 1975). This theory was confirmed when thiomolybdatecompounds were given to rats and produced anti-copper effects (Mills etal., 1981a, b; Bremner et al., 1982). The tetrathio-substitutedcompound, TM, is the most potent of these.

The anti-copper mechanism of action of TM is two fold (Mills et al.,1981a, b; Bremner et al., 1982; Gooneratne et al., 1981b). One mechanismoperates in the GI tract, the second in the blood. In the GI tract, TMforms complexes with copper and food proteins (or other proteins), thatare not absorbed. This absorption block involves not only food copper,but also the rather considerable amount of endogenously secreted copperin saliva, gastric juice and other GI tract secretions (Allen andSolomons, 1984). TM is a more effective blocker of copper absorptionthan zinc, since zinc acts only in those areas. of the small intestinewhere metallothionein can be induced (Yuzbasiyan-Gurkan et al., 1992).In contrast, TM works all up and down the GI tract. The other advantageof TM over zinc in this setting is that TM acts immediately. It does nothave a lag period required for the induction of metallothionein.

The second effect of TM is on the blood. TM given at times away frommeals is relatively well absorbed into the blood. There it formscomplexes with copper and albumin, rendering the complexed copperunavailable for cellular uptake (Gooneratne et al., 1981b). The normalplasma copper is in two primary pools. Most of the plasma copper innormal persons is part of the ceruloplasmin molecule. This copper isessentially unavailable for ready exchange with cells and is considerednon-toxic. The other pool of copper is more loosely bound to albumin andsmall molecules, such as amino acids. This pool of copper is greatlyexpanded during acute copper toxicity in diseases such as Wilson'sdisease, and is readily available for cellular uptake and is, therefore,potentially toxic (Scheinberg and Sternlieb, 1984). When TM enters theblood it complexes with this latter copper and renders it, like theceruloplasmin copper, unavailable for cellular uptake and for furthertoxicity.

Very good evidence exists that TM-complexed copper is unavailable forcellular uptake. The most direct evidence is that in sheep levels ofcopper in the plasma which would normally be high enough to producehemolytic anemia do not do so in the presence of TM (Gooneratne et al.,1981b). It was shown that the TM bound copper does not permeate theerythrocyte. This is direct evidence that TM-complexed copper does notpermeate cells.

TM and salts of TM are available; one of the preferred salts of TM isthe ammonium salt. TM as purchased from Aldrich Chemical Company(catalog number W180-0; Milwaukee, Wis.), is a black powder that ismoderately water soluble, yielding a bright red solution. TM purchasedfrom Aldrich Chemical Company (available in one kilogram bulk lots) iscertified pure for human use. The bulk drug should be stored in theabsence of oxygen, or the oxygen will gradually exchange with thesulfur, rendering the drug ineffective over time. The bulk drug istherefore stored under argon. Stability assays developed by theinventors indicate that the drug is stable for several years under argon(Brewer et al., 1991a). Capsules can be filled by hand, and the drug isstable in capsules for several months at room temperature.

TM acts by forming a tripartite complex with copper and protein (Millset al., 1981a, b; Bremner et al., 1982). TM has two mechanisms ofaction. Given with meals, it complexes copper in food and endogenouslysecreted copper with itself and food protein, and prevents theabsorption of copper. Patients can be put into an immediate negativecopper balance with TM by administering it with meals. Given betweenmeals, the TM is absorbed into the bloodstream, and complexes serumcopper with itself and albumin, rapidly rendering the copper unavailablefor cellular uptake. Since free copper in organs is in equilibrium withfree copper of blood, free copper in the organs, and in tumor tissue,will quickly be reduced to very low levels, if the blood copper isbound. This complex is cleared through the kidney and the liver. TM isthe most potent and most rapidly acting anti-copper agent known.

2. TM Toxicity and Efficacy

Tetrathiomolybdate (TM) is a drug that the inventors have developed asan orphan therapy for Wilson's disease. The drug does an excellent jobof gaining quick control over copper toxicity and preventing theneurological worsening that occurs 50% of the time during initialtreatment with a commonly used drug for Wilson's disease, penicillamine(Brewer et al., 1991a; Brewer et al., 1994b; Brewer et al., 1996). Sofar, the inventors have treated 55 Wilson's disease patients with TM,generally for an eight week period. TM thus fills a very important nichein the initial treatment of Wilson's disease. The Wilson's disease workhas provided extensive experience with TM in the human, and has helpedto document TM's extremely low level of toxicity in humans.

In human Wilson's disease studies, the one side effect occasionallyobserved is a reversible anemia, due to TM's anti-copper effects. Givenin too high a dose, TM renders the bone marrow severely or totallycopper deficient. Since copper is required for erythropoiesis, an anemiadevelops. That anemia is rapidly reversible by simply stopping TM. Inthe Wilson's disease studies, the overtreatment effect of TM has beendiminished by simply reducing the dose to 20 mg six times per day. Inhumans without Wilson's disease, such as cancer patients, a level ofmild copper deficiency at a pre-anemia state can be established simplyby carefully monitoring ceruloplasmin (Cp) levels during TM therapy.

TM is eventually metabolized to thiomolybdates, molybdates andmolybdenum oxides, so the potential toxicity of these compounds have tobe considered. However, it turns out that these molybdenum compounds arequite innocuous at the levels produced from breakdown of TM used in theclinical situations described herein. About 37% of TM by weight is Mo,so in the studies described herein, up to 50 mg of Mo/day isadministered for two weeks then no more than about 25 mg/day formaintenance. High doses of 350 to 1400 mg/day of Mo were previously usedfor 4-11 months in patients with Wilson's disease, without toxicity(Bickel et al., 1957). Thus, the dose range of 25-50 mg/day poses nopredictable problems, and should be entirely safe.

Considerable work on the potential toxicity of TM has been carried outin rats (Mills et al., 1981a; Bremner et al., 1982). At approximately 6mg of TM per kilogram of diet rats show substantial effects on copper,including a reduction of plasma ceruloplasmin and a reduction in liverand kidney copper. At approximately 12 mg of TM all of these changeswere increased and, in addition, liver Mo was increased. Mild anemia waspresent, and skeletal lesions were present in one of six animals. At 18mg of TM the anemia was severe. Melanogenesis of hair was impaired,diarrhea was present, growth rate was markedly impaired, and all animalshad skeletal lesions characterized by dysplasia in the epiphysealcartilage cells of long bones, resorption of trabecular bone, andstructural changes in ligaments.

It was later shown that all of the toxic effects of TM, up to 36 mg ofTM per kilogram of diet, could be prevented by oral supplementation withcopper, or with intraperitoneal injection of copper (Mills et al.,1981b). Thus, it appears that all the toxic lesions induced by TM aredue to copper deficiency induced by the TM. In support of this, almostall of the above lesions are induced by dietary copper deficiency, thetwo exceptions being the skeletal lesions and the enterocytemitochondrial damage which leads to diarrhea. The reason that these lasttwo lesions are seen with TM administration, but may not be seen indietary copper deficiency, could be related to the severity and therapidity of the copper deficiency induced by TM. With dietary copperdeficiency there is always some contaminating copper available, andrapidly dividing cells such as the enterocyte and epiphyseal cells mayobtain enough copper to prevent the lesions. The prevention of these twolesions as well as all of the other TM induced lesions by coppersupplementation indicates that the lesions are probably due to copperdeficiency.

Another group has examined gut pathology in rats receiving approximately18 mg of TM per kilogram of diet (Fell et al., 1979). These rats alsoreceived approximately 3 mg of copper per kilogram of diet. Thesescientist found gut pathology involving cell apoptosis, edema, andnecrosis which they did not attribute to hypocuprosis, although this wasnot proven. It is probable that a higher copper supplement was requiredfor protection, in view of the finding that all such problems wereprevented by adequate copper supplementation (Mills et al., 1981b).

Wilson's disease patients have a huge store of excess copper, so none ofthe TM toxicities due to copper deficiency are a risk in these patients.Even in the case of the skeletal and enterocyte lesions, since copperadministration protected, the Wilson's disease patient with excessivestores of copper should also be protected. Other workers have studiedthe effect of TM on copper loaded sheep (Gooneratne et al., 1981a). Itis well known that sheep are quite susceptible to copper toxicity,usually developing hepatic failure and hemolytic anemia. The studiesinvolved loading sheep dietarily with copper to the point of initiationof hepatic damage, then TM was given intravenously in doses of 50 or 100mg 2× weekly for up to 11 weeks.

Five of the 26 sheep died during the study. All deaths were attributedto copper toxicosis based on autopsy results. 3 of the 5 deaths occurredin controls that received copper but not TM. One death occurred after ananimal had received only one dose of TM, and another in a animal thathad received only 4 doses. It is clear that these 2 animals died fromcopper toxicity prior to the ability of TM to rescue them. If animalssurvived the initial onset of copper toxicosis, they were protected fromfurther copper toxicity by TM, even though in some cases copperadministration was continued. These animals tolerated up to 22injections of TM without clinical problems.

Support for the beneficial effect of either IV (Humphries et al., 1986)or subcutaneous (Humphries et al., 1988) TM in protecting sheep againstsevere hepatic copper toxicity has also been shown. TM not only reducedthe amount of hepatic copper, but the actual liver damage. TM was alsoused prophylactically to prevent copper toxicity in commercial sheepflocks. Over 400 animals have been treated with TM with no adverse sideeffects (Humphries et al., 1988).

Preliminary work also indicated that TM may be dramatically effectiveagainst copper toxicity in the LEC rat model (Suzuki et al., 1993). Thegenetic defect on these rats has been recently shown to be due to adefect in the Wilson's disease gene (Wu et al., 1994). The rats developsevere liver disease and usually die. TM has been very effective intreating these animals in the late stages of their liver disease.

Mo metabolism in sheep has been studied after the IV injection of ⁹⁹Molabeled TM (Mason et al., 1983). There was a rapid plasma disappearanceover 15 minutes and then a slow disappearance with a t_(1/2) of about 40h. The TM was transformed step wise to molybdate and over 90% wasexcreted in urine compared to 5% in feces. The same group publishedsubsequently on ⁹⁹Mo and ³⁵S metabolism after IV injection of doublelabeled TM in sheep (Hynes et al., 1984). Most of the ⁹⁹Mo and ³⁵S wereassociated initially with albumin. Displaced or unbound TM was rapidlyhydrolyzed to molybdate and sulfate. There was no evidence of anirreversible interaction of either ³⁵S or ⁹⁹Mo with copper and plasmadespite the appearance of a TCA insoluble copper fraction.

It is clear that in the presence of high levels of copper, TMadministration results in the accumulation of copper complexed with TMin both the liver and kidneys (Jones et al., 1984; Bremner and Young,1978). However, there is no evidence of a storage disease associatedwith this complex. Current theory holds that the complex isdisassociated and that the TM is metabolized to oxymolybdates andexcreted (Mason et al., 1983). The copper then enters other pathways inthe liver. In the presence of high levels of metallothionein it wouldmost likely be taken up by metallothionein. In the kidneys the evidenceis that the copper is simply excreted.

Two cases of reversible bone marrow depression have been reported inpatients receiving TM for maintenance therapy (Harper and Walshe, 1986).The inventors have seen reversible anemia in seven patients. Thesepatients had a strong response to therapy, and likely ended up withlocalized, bone marrow, copper deficiency. Since copper is required forheme synthesis, this appears to be a manifestation of over-treatment, atleast as far as the bone marrow is concerned. Since TM is such aneffective anti-copper agent, during maintenance therapy with TM as inthe cases of Harper and Walshe (Harper and Walshe, 1986), it would notbe unexpected for over-treatment to occur.

3. Molybdenum (Mo) Toxicity

About 37% of TM is Mo. The normal intake of Mo is about 350 μg/day(Seelig, 1972), or the equivalent amount of Mo that would be in about1.0 mg of TM. Molybdenum seems to be quite well tolerated by the human.Relatively high doses of 5-20 mg/kg/day of Mo (equivalent to the Mo in1-4 g of TM) were used for 4-11 months in patients with Wilson's diseasein a 1957 study, without known toxicity (Bickel et al., 1957). However,it was not effective, because as pointed out earlier, TM is the activemetabolite, and that is formed efficiently from Mo only in ruminants.

B. Other Thiomolybdate Compounds

Other thiomolybdate compounds that have a similar mode of action in freecopper reduction include dodecathiodimolybdate, trithiomolybdate,dithiomolybdate and monothiomolybdate. These compounds, liketetrathiomolybdate, form a tripartite complex with copper and proteinthat renders the copper unavailable, and eventually leads to clearanceof the copper-complex.

The synthesis and characterization of a number of thiomolybdatecomplexes, oxo/thiomolybdate complexes and heterometallic complexescontaining iron, molybdenum and sulfur, with or without oxygen, havebeen described (Coucouvanis, 1998; Coucouvanis et al., 1989; Coucouvaniset al., 1988; Hadjikyriacou and Coucouvanis, 1987; Coucouvanis et al.,1984; Teo et al., 1983; Coucouvanis et al., 1983; Kanatzidis andCoucouvanis, 1983; Coucouvanis et al., 1981; Coucouvanis, 1981;Coucouvanis et al., 1980a, b). These complexes have been shown to bindto copper, and are thus contemplated for use in certain embodiments ofthe invention.

Among the preferred thiomolybdate compounds or complexes are thosecomprising one, two or three molybdenum atom(s), one molybdenum atom andone iron atom, two molybdenum atoms and one iron atom, and onemolybdenum atom and two iron atoms. The structures of three generalclasses of thiomolybdate compounds, and two of the preferredthiomolybdate compounds, are shown below:

where Cat is a cation that makes the complex water soluble, for exampleNH₄ ⁺, L is a ligand such as Cl⁻ or R-Ph-S, where R is a functionalgroup that makes the complex water soluble, such as a sulfonate, and Phis a phenyl group.

Examples of the thiomolybdate complexes contemplated for use in thepresent invention include, but are not limited to, nonathiomolybdate([MoS₉]²⁻), hexathiodimolybdate ([Mo₂S₆]²⁻), heptathiodimolybdate([Mo₂S₇]²⁻), octathiodimolybdate ([Mo₂S₈]²⁻), nonathiodimolybdate([Mo₂S₉]²⁻), decathiodimolybdate ([Mo₂S₁₂]²⁻), undecathiodimolybdate([Mo₂S₁₁]²⁻), dodecathiodimolybdate ([Mo₂S₁₂]²⁻), the ammonium salt ofdodecathiodimolybdate ((NH₄)₂[Mo₂(S₂)₆]; Muller et al., 1980; Muller andKrickemeyer, 1990), the ammonium salt of tridecathiotrimolybdate((NH₄)₂[Mo₃(S)(S₂)₆]; Muller and Krickemeyer, 1990), and thosethiomolybdates with an [FeCl₂S₂MoS₂FeCl₂]²⁻, [S₂MoS₂FeS₂MoS₂]³⁻, and[S₂MoS₂FeCl₂]²⁻ core structures.

Additionally, the inventors have discovered that iron gluconate is asource of iron that does not contain halide ions. Its reaction withtetrathiomolybdate in the presence of ammonium hydroxide producesanother preferred thiomolybdate compound, the novel ammonium salt ofiron octathiodimolybdate ((NH₄)₃[S₂MoS₂FeS₂MoS₂]), which is very watersoluble.

While certain of these compounds may be less potent in reducing copperlevels in the body than tetrathiomolybdate, they will nonetheless findutility, and in particular instances be preferred, in certain aspects ofthe present invention.

C. Stabilized Thiomolybdate Complexes

Thiomolybdate complexes are sensitive to oxidation when exposed to air.The inventors have discovered that by forming a complex betweenthiomolybdate compounds and carbohydrates, such as sucrose, that astabilized form of the thiomolybdate compounds are produced. In thesethiomolybdate-carbohydrate complexes, layers of carbohydrate moleculesassemble around the thiomolybdate compound in arrays stabilized byhydrogen bonding. These layers serve to protect the thiomolybdate coreagainst oxidation and hydrolysis. Other molecules, such as amino acids,that are capable of hydrogen bonding to the thiomolybdate compounds, arealso contemplated for use in certain aspects of the invention.

The term “carbohydrate” embraces a wide variety of chemical compoundshaving the general formula (CH₂O)_(n) and encompasses such compounds asmonosaccharides, disaccharides, trisaccharides, oligosaccharides,polysaccharides and their aminated, sulfated, acetylated and otherderivated forms. Oligosaccharides are chains composed of sugar units,which are also known as monosaccharides. Sugar units can be arranged inany order and linked by their sugar units in any number of differentways. Therefore, the number of different stereoisomeric oligosaccharidechains possible is quite large.

As known in the art, a monosaccharide is a sugar molecule that containsone sugar unit. As used herein, the term “sugar unit” means amonosaccharide. As also known in the art, a disaccharide is a sugarmolecule that contains 2 sugar units, a trisaccharide is a sugarmolecule that contains 3 sugar units, an oligosaccharide is a sugarmolecule that generally contains between about 2 and about 10 sugarunits, and a polysaccharide is a sugar molecule that contains greaterthan 10 sugar units. The sugar units in a di-, tri- and oligosaccharideare all connected by glycosidic linkages. Nonetheless, as used in thepresent invention, the term “oligosaccharide” means a sugar moleculethat contains at least two sugar units.

Within the scope of the present invention, “monosaccharides” will beunderstood as including, but not being limited to, either the D- orL-isomers of trioses, aldopentoses, aldohexoses, aldotetroses,ketopentoses and ketohexoses. The mentioned compounds may also be in theform of lactones. Examples of an aldopentose include, but are notlimited to, ribose, arabinose, xylose and lyose; examples of analdohexose are allose, altrose, glucose, mannose, gulose, idose,galactose, talose, fucose and rhamnose. Examples of a ketopentoseinclude, but are not limited to, ribulose and xylulose, examples of atetrose include, but are not limited to, erythrose and threose, andexamples of a ketohexose include, but are not limited to, psicose,fructose, sorbose or tagatose.

Examples of a disaccharide are trehalose, maltose, isomaltose,cellobiose, gentiobiose, saccharose, lactose, chitobiose,N,N-diacetylchitobiose, palatinose or sucrose. Examples oftrisaccharides are raffinose, panose, melezitose or maltotriose.Examples of oligosaccharides are maltotetraose, maltohexaose orchitoheptaose. The term “carbohydrate” as used herein is also intendedto embrace sugar alcohols, e.g., alditols such as mannitol, lactitol,xylitol, glycerol or sorbitol. Examples of polysaccharides include, butare not limited to, polydextrose and maltodextrin.

This stabilization is contemplated to be effective using anycarbohydrate as defined herein, at ratios of between about 5 sugar unitsto about 100 or 200 sugar units or so per thiomolybdate metal center.This range will be understood to include all values within this range,such as ratios of about 10 sugar units per thiomolybdate metal center,about 20 sugar units per thiomolybdate metal center, about 25 sugarunits per thiomolybdate metal center, about 30 sugar units perthiomolybdate metal center, about 40 sugar units per thiomolybdate metalcenter, about 50 sugar units per thiomolybdate metal center, about 60sugar units per thiomolybdate metal center, about 70 sugar units perthiomolybdate metal center, about 75 sugar units per thiomolybdate metalcenter, about 80 sugar units per thiomolybdate metal center, about 90sugar units per thiomolybdate metal center, about 110 sugar units perthiomolybdate metal center, about 125 sugar units per thiomolybdatemetal center, about 150 sugar units per thiomolybdate metal center,about 175 sugar units per thiomolybdate metal center or about 190 sugarunits per thiomolybdate metal center.

An example of such a stabilized thiomolybdate compound istetrathiomolybdate stabilized by sucrose. To prepare thesucrose-ammonium tetrathiomolybdate complex, 25 grams of sucrose isdissolved in 20 ml of distilled water. 1 gram of ammoniumtetrathiomolybdate (TM) is added and the mixture stirred under argonuntil the TM is solution. The water is then removed from the mixture byflash evaporation under high vacuum.

The resulting powder appears to be much more stable in open air than thepure TM. After 3 months of storage in an open dish, the sucrose powderretained 61% of the original drug as measured by the OD₄₆₇, while the TMstored under the same conditions retained only 5% of its original OD₄₆₇.

D. Monitoring Copper Levels

The serum ceruloplasmin, which is directly dependent upon liver copperstatus, is an accurate indicator of copper status. The dose of TM usedin previous studies in rodents would average about 0.5 mg/day in rats ofaverage weight. The inventors' studies in mice and rats, and theinventors' extensive experience in humans with Wilson's disease and inhumans with cancer, indicate that 4 times that dose is required in ratsto reduce serum ceruloplasmin to about 10% of normal which is theoptimal criterion for low copper status in rodents. These concepts ofoptimal monitoring of Cu status and TM dosing were subsequentlyincorporated in the animal studies of TM described in Example 2.

Based on the studies herein, and the inventors experience with TM,reduction in the level of ceruloplasmin (Cp) to between about 40% andabout 10% of the baseline value prior to treatment will result in atleast some level of beneficial clinical anti-angiogenic effect. However,a reduction of Cp to a level of about 20% of the baseline value prior totreatment is preferred in most clinical indications.

II. Other Copper Chelating Agents

In certain aspects of the present invention, the inventors contemplatethe use of zinc to achieve a low-copper status in cancer patients. Theinventors have developed zinc as an anti-copper agent for Wilson'sdisease, and it was approved for this purpose in January 1997 by theUnited States Food and Drug Administration (FDA). Zinc acts by inducingmetallothionein in the intestinal mucosal cell, thereby blocking copperabsorption (Brewer and Yuzbasiyan-Gurkan, 1992a). Zinc too, is extremelysafe. In studies now reaching 200 Wilson's disease patients, zinc indoses of 150 mg daily has produced no toxicity at all (Brewer andYuzbasiyan-Gurkan, 1992a).

In general, zinc lowers copper levels more slowly thantetrathiomolybdate and other thiomolybdate compounds. However, due tothe fact that zinc compounds are relatively inexpensive and easy toprepare, and their widespread availability, the use of zinc compounds ispreferred in certain aspects of the present invention, both in achievinginitial low-copper status, and in maintenance therapy once low-copperstatus has been achieved using other agents (discussed in greater detailbelow).

III. Combination Therapy

The methods of the present invention may be combined with any othermethods generally employed in the treatment of the particular disease ordisorder that the patient exhibits. For example, in connection with thetreatment of solid tumors, the methods of the present invention may beused in combination with classical approaches, such as surgery,radiotherapy and the like. So long as a particular therapeutic approachis not known to be detrimental in itself, or counteracts theeffectiveness of the TM therapy, its combination with the presentinvention is contemplated. When one or more agents are used incombination with the TM therapy, there is no requirement for thecombined results to be additive of the effects observed when eachtreatment is conducted separately, although this is evidently desirable,and there is no particular requirement for the combined treatment toexhibit synergistic effects, although this is certainly possible andadvantageous.

In terms of surgery, concurrent surgery during copper deficiency is notpreferred since blood vessel growth is required for wound healing.However, as copper can be repleted within about 24 hours ofdiscontinuing TM therapy, any surgical intervention may be practicedafter copper repletion. TM therapy can then be reinstated after woundhealing has been established, typically about 1-2 weeks followingsurgery. In connection with radiotherapy, any mechanism for inducing DNAdamage locally within tumor cells is contemplated, such asγ-irradiation, X-rays, UV-irradiation, microwaves and even electronicemissions and the like. The directed delivery of radioisotopes to tumorcells is also contemplated, and this may be used in connection with atargeting antibody or other targeting means.

Cytokine therapy also has proven to be an effective partner for combinedtherapeutic regimens. Various cytokines may be employed in such combinedapproaches. Examples of cytokines include IL-1α IL1β, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, TGF-β, GM-CSF,M-CSF, G-CSF, TNFα, TNFβ, LAF, TCGF, BCGF, TRF, BAF, BDG, MP, LIF, OSM,TMF, PDGF, IFN-α, IFN-β, IFN-γ. Cytokines are administered according tostandard regimens, consistent with clinical indications such as thecondition of the patient and relative toxicity of the cytokine.

A. Copper Chelating Agents

The inventors contemplate the use of zinc following achievement of alow-copper status in cancer patients in order to maintain a moderatecopper-deficient state for the long-term. As discussed above, theinventors have developed zinc as an anti-copper agent for Wilson'sdisease, and it was approved for this purpose in January 1997 by theUnited States Food and Drug Administration (FDA). Zinc acts by inducingmetallothionein in the intestinal mucosal cell, thereby blocking copperabsorption (Brewer and Yuzbasiyan-Gurkan, 1992a). Zinc too, is extremelysafe. In studies now reaching 200 Wilson's disease patients, zinc indoses of 150 mg daily has produced no toxicity at all (Brewer andYuzbasiyan-Gurkan, 1992a).

1. Zinc

Zinc compounds, such as zinc acetate, are being used for thecomprehensive treatment of Wilson's disease including initial treatment(Hoogenraad et al., 1978; Hoogenraad et al., 1979; Hoogenraad et al.,1987). However, zinc is not ideal for initial therapy (by itself)because it is rather slow acting. Thus, it takes approximately two weeksto achieve intestinal metallothionein induction and a negative copperbalance (Yuzbasiyan-Gurkan et al., 1992). At the two week point, zincimmediately reverses the +0.54 mg daily (positive) copper balance thesepatients average, but the negative copper balance induced is rathermodest, averaging −0.35 mg daily (negative) copper balance (Brewer etal., 1990; Brewer et al., 1993b). Due to this low rate of copperremoval, it takes as long as six months of zinc therapy to bring urinecopper and nonceruloplasmin plasma copper (the potentially toxic coppermeasured in the blood), down to subtoxic levels.

2. Penicillamine

Penicillamine is the drug that has been used the most, and is the bestknown. However, it should be the last choice for initial treatment ofneurological patients because of the very high risk of making themneurologically worse (Brewer et al., 1987a; Glass et al., 1990; Breweret al., 1994a). Another problem with penicillamine is that about aquarter to a third of patients develop an initial hypersensitivitysyndrome, requiring significant interventions, such as temporarilystopping the drug and restarting it at a lower dose, usually withconcurrent cortico-steroid administration. This is a somewhatfrightening experience for patients who are already ill, and preventsthe attending physician in the inventors' study from being blinded.Finally, there is a long list of other side effects that can occur withpenicillamine during the first few weeks of therapy. These include bonemarrow depression, proteinuria, and auto-immune disorders.

3. Trientine

Trientine acts by chelation and urinary excretion of copper (Walshe,1982). A therapeutic dose (1,000-2,000 mg/day) usually produces onlyabout half as much cupruresis as a similar dose of penicillamine.Nonetheless, trientine is capable of an initial production of a severalmg negative copper balance, much greater than zinc. Typically, this 4-5mg cupruresis decreases during the first few weeks of therapy to a moremodest, but still substantial, 2-3 mg. Ingestion of copper is about 1mg/day, with obligatory, non-urine losses of about 0.5 mg. Thus acupruresis of 2-3 mg produces a negative copper balance of 1.5 to 2.5mg/day.

Trientine is officially approved for use in patients intolerant ofpenicillamine therapy. Because of this, and because it was introducedmuch later than penicillamine, it has not been used and reported on veryextensively. It has not had a formal toxicity study. It appears to havesubstantially less risk of side effects then penicillamine. An initialhypersensitivity problem has not been reported. It does causeproteinuria, after several weeks of use in about 20% of patients. It canalso occasionally produce bone marrow depression and autoimmuneabnormalities, although the latter is usually after prolonged use.

So far, trientine has not been reported to cause initial worsening inneurological patients, but its sole use in this type of patient isprobably very limited. Anecdotally, the inventors have received patientsin transfer who worsened on penicillamine, were switched briefly totrientine, and when they became worse (or failed to improve) weretransferred to the inventors for TM therapy. In these kinds of patientsit is impossible to know if trientine played any role in worsening.Theoretically, it could, because as with penicillamine, it mobilizescopper, producing a higher blood level to achieve urinary excretion. Butwhether this increased level of blood copper translates into increasedbrain levels, and increased neurotoxicity, is unknown.

B. Chemotherapeutic Combinations and Treatment

Irrespective of the mechanisms by which enhanced tumor destruction isachieved, the combined treatment aspects of the present invention haveevident utility in the effective treatment of disease. To use thepresent invention in combination with the administration of achemotherapeutic agent, one would simply administer to an animal anagent that binds copper and forms a tripartite agent-copper-proteincomplex in combination with the chemotherapeutic agent in a mannereffective to result in their combined anti-tumor actions within theanimal. These agents would therefore be provided in an amount effectiveand for a period of time effective to result in their combined presencewithin the tumor vasculature and their combined actions in the tumorenvironment. To achieve this goal, the agent that binds copper and formsa tripartite agent-copper-protein complex and chemotherapeutic agent maybe administered to the animal simultaneously, either in a singlecomposition or as two distinct compositions using differentadministration routes.

Alternatively, treatment with an agent that binds copper and forms atripartite agent-copper-protein complex may precede or follow thechemotherapeutic agent treatment by intervals ranging from minutes toweeks. In embodiments where the chemotherapeutic factor and agent thatbinds copper and forms a tripartite agent-copper-protein complex areapplied separately to the animal, one would generally ensure that asignificant period of time did not expire between the time of eachdelivery, such that the chemotherapeutic agent and agent that bindscopper and forms a tripartite agent-copper-protein complex would stillbe able to exert an advantageously combined effect on the tumor. In suchinstances, it is contemplated that one would contact the tumor with bothagents within about 5 minutes to about one week of each other and, morepreferably, within about 12-72 hours of each other, with a delay time ofonly about 12-48 hours being most preferred.

However, in some situations, it may be desirable to extend the timeperiod for treatment significantly, where several days (2, 3, 4, 5, 6 or7) or even several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between therespective administrations. Additionally, as described above, apreferred embodiment of the present invention is to administer the agentthat binds copper and forms a tripartite agent-copper-protein complexbetween rounds of chemotherapy, or in a maintenance regimen afterchemotherapy. Thus, it also is conceivable that more than oneadministration of either the agent that binds copper and forms atripartite agent-copper-protein complex and/or the chemotherapeuticagent will be desired. To achieve tumor regression, both agents aredelivered in a combined amount effective to inhibit its growth,irrespective of the times for administration.

A variety of chemotherapeutic agents are intended to be of use in thecombined treatment methods disclosed herein. Chemotherapeutic agentscontemplated as exemplary include, e.g., etoposide (VP-16), adriamycin,5-fluorouracil (5FU), camptothecin, actinomycin-D, mitomycin C,carbaplatin, paclitaxel, docetaxel and even hydrogen peroxide. As willbe understood by those of ordinary skill in the art, the appropriatedoses of chemotherapeutic agents will be generally around those alreadyemployed in clinical therapies wherein the chemotherapeutics areadministered alone or in combination with other chemotherapeutics.

Further useful agents include compounds that interfere with DNAreplication, mitosis and chromosomal segregation. Such chemotherapeuticcompounds include adriamycin, also known as doxorubicin, etoposide,verapamil, podophyllotoxin, and the like. Widely used in a clinicalsetting for the treatment of neoplasms, these compounds are administeredthrough bolus injections intravenously at doses ranging from 25-75 mg/m²at 21 day intervals for adriamycin, to 35-50 mg/m² for etoposideintravenously or double the intravenous dose orally.

Agents that disrupt the synthesis and fidelity of polynucleotideprecursors may also be used. Particularly useful are agents that haveundergone extensive testing and are readily available. As such, agentssuch as 5-fluorouracil (5-FU) are preferentially used by neoplastictissue, making this agent particularly useful for targeting toneoplastic cells. Although quite toxic, 5-FU, is applicable in a widerange of carriers, including topical, however intravenous administrationwith doses ranging from 3 to 15 mg/kg/day being commonly used.

Exemplary chemotherapeutic agents that are useful in connection withcombined therapy are listed in Table 1. Each of the agents listedtherein are exemplary and by no means limiting. The skilled artisan isdirected to “Remington's Pharmaceutical Sciences” 15th Edition, chapter33, in particular pages 624-652. Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject. Moreover, forhuman administration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiologics standards.

TABLE 1 Chemotherapeutic Agents Useful In Neoplastic DiseaseNONPROPRIETARY NAMES CLASS TYPE OF AGENT (OTHER NAMES) DISEASEAlkylating Nitrogen Mustards Mechlorethamine (HN₂) Hodgkin's disease,non-Hodgkin's Agents lymphomas Cyclophosphamide Acute and chroniclymphocytic Ifosfamide leukemias, Hodgkin's disease, non- Hodgkin'slymphomas, multiple myeloma, neuroblastoma, breast, ovary, lung, Wilms'tumor, cervix, testis, soft-tissue sarcomas Melphalan (L-sarcolysin)Multiple myeloma, breast, ovary Chlorambucil Chronic lymphocyticleukemia, primary macroglobulinemia, Hodgkin's disease, non-Hodgkin'slymphomas Ethylenimenes and Hexamethylmelamine Ovary MethylmelaminesThiotepa Bladder, breast, ovary Alkyl Sulfonates Busulfan Chronicgranulocytic leukemia Nitrosoureas Carmustine (BCNU) Hodgkin's disease,non-Hodgkin's lymphomas, primary brain tumors, multiple myeloma,malignant melanoma Lomustine (CCNU) Hodgkin's disease, non-Hodgkin'slymphomas, primary brain tumors, small-cell lung Semustine (methyl-CCNU)Primary brain tumors, stomach, colon Streptozocin Malignant pancreaticinsulinoma, (streptozotocin) malignant carcinoid Triazines Dacarbazine(DTIC; Malignant melanoma, Hodgkin'sdimethyltriazenoimidazolecarboxamide) disease, soft-tissue sarcomasAntimetabolites Folic Acid Analogs Methotrexate Acute lymphocyticleukemia, (amethopterin) choriocarcinoma, mycosis fungoides, breast,head and neck, lung, osteogenic sarcoma Pyrimidine Analogs Fluouracil(5-fluorouracil; Breast, colon, stomach, pancreas, 5-FU) ovary, head andneck, urinary Floxuridine (fluorodeoxyuridine; bladder, premalignantskin lesions FUdR) (topical) Cytarabine (cytosine Acute granulocytic andacute arabinoside) lymphocytic leukemias Purine Analogs andMercaptopurine Acute lymphocytic, acute Related Inhibitors(6-mercaptopurine; granulocytic and chronic 6-MP) granulocytic leukemiasThioguanine Acute granulocytic, acute (6-thioguanine; TG) lymphocyticand chronic granulocytic leukemias Pentostatin Hairy cell leukemia,mycosis (2-deoxycoformycin) fungoides, chronic lymphocytic leukemiaNatural Vinca Alkaloids Vinblastine (VLB) Hodgkin's disease,non-Hodgkin's Products lymphomas, breast, testis Vincristine Acutelymphocytic leukemia, neuroblastoma, Wilms' tumor, rhabdomyosarcoma,Hodgkin's disease, non-Hodgkin's lymphomas, small-cell lungEpipodophyllotoxins Etoposide Testis, small-cell lung and other lung,Tertiposide breast, Hodgkin's disease, non- Hodgkin's lymphomas, acutegranulocytic leukemia, Kaposi's sarcoma Antibiotics DactinomycinChoriocarcinoma, Wilms' tumor, (actinomycin D) rhabdomyosarcoma, testis,Kaposi's sarcoma Daunorubicin Acute granulocytic and acute (daunomycin;lymphocytic leukemias rubidomycin) Doxorubicin Soft-tissue, osteogenicand other sarcomas; Hodgkin's disease, non- Hodgkin's lymphomas, acuteleukemias, breast, genitourinary, thyroid, lung, stomach, neuroblastomaBleomycin Testis, head and neck, skin, esophagus, lung and genitourinarytract; Hodgkin's disease, non- Hodgkin's lymphomas Plicamycin(mithramycin) Testis, malignant hypercalcemia Mitomycin (mitomycin C)Stomach, cervix, colon, breast, pancreas, bladder, head and neck EnzymesL-Asparaginase Acute lymphocytic leukemia Biological Response Interferonalfa Hairy cell leukemia., Kaposi's Modifiers sarcoma, melanoma,carcinoid, renal cell, ovary, bladder, non-Hodgkin's lymphomas, mycosisfungoides, multiple myeloma, chronic granulocytic leukemia MiscellaneousPlatinum Cisplatin (cis-DDP) Testis, ovary, bladder, head and AgentsCoordination Carboplatin neck, lung, thyroid, cervix, Complexesendometrium, neuroblastoma, osteogenic sarcoma AnthracenedioneMitoxantrone Acute granulocytic leukemia, breast Substituted UreaHydroxyurea Chronic granulocytic leukemia, polycythemia vera, essentalthrombocytosis, malignant melanoma Methyl Hydrazine ProcarbazineHodgkin's disease Derivative (N-methylhydrazine, MIH) AdrenocorticalMitotane (o,p'-DDD) Adrenal cortex Suppressant Aminoglutethimide BreastHormones and Adrenocorticosteroids Prednisone (several other Acute andchronic lymphocytic Antagonists equivalent preparations leukemias,non-Hodgkin's available) lymphomas, Hodgkin's disease, breast ProgestinsHydroxyprogesterone Endometrium, breast caproate Medroxyprogesteroneacetate Megestrol acetate Estrogens Diethylstilbestrol Breast, prostateEthinyl estradiol (other preparations available) Antiestrogen TamoxifenBreast Androgens Testosterone propionate Breast Fluoxymesterone (otherpreparations available) Antiandrogen Flutamide Prostate Gonadotropin-Leuprolide Prostate releasing hormone analog

C. Anti-Angiogenics

The term “angiogenesis” refers to the generation of new blood vessels,generally into a tissue or organ. Under normal physiological conditions,humans or animals undergo angiogenesis only in very specific restrictedsituations. For example, angiogenesis is normally observed in woundhealing, fetal and embryonic development and formation of the corpusluteum, endometrium and placenta.

Both controlled and uncontrolled angiogenesis are thought to proceed ina similar manner. Endothelial cells and pericytes, surrounded by abasement membrane, form capillary blood vessels. Angiogenesis beginswith the erosion of the basement membrane by enzymes released byendothelial cells and leukocytes. The endothelial cells, which line thelumen of blood vessels, then protrude through the basement membrane.Angiogenic stimulants induce the endothelial cells to migrate throughthe eroded basement membrane. The migrating cells form a “sprout” offthe parent blood vessel, where the endothelial cells undergo mitosis andproliferate. The endothelial sprouts merge with each other to formcapillary loops, creating the new blood vessel.

Persistent, unregulated angiogenesis occurs during tumor metastasis. Theagents of the invention that bind copper and form a tripartiteagent-copper-protein complex may thus be used in combination withadditional “anti-angiogenic” therapies. A preferred component for use ininhibiting angiogenesis is a protein named “angiostatin”. This componentis disclosed in U.S. Pat. Nos. 5,776,704; 5,639,725 and 5,733,876, eachincorporated herein by reference. Angiostatin is a protein having amolecular weight of between about 38 kDa and about 45 kDa, as determinedby reducing polyacrylamide gel electrophoresis, which containsapproximately Kringle regions 1 through 4 of a plasminogen molecule.Angiostatin generally has an amino acid sequence substantially similarto that of a fragment of murine plasminogen beginning at amino acidnumber 98 of an intact murine plasminogen molecule.

The amino acid sequence of angiostatin varies slightly between species.For example, in human angiostatin, the amino acid sequence issubstantially similar to the sequence of the above described murineplasminogen fragment, although an active human angiostatin sequence maystart at either amino acid number 97 or 99 of an intact humanplasminogen amino acid sequence. Further, human plasminogen may be used,as it has similar anti-angiogenic activity, as shown in a mouse tumormodel.

Specific angiogenesis inhibitors, including, but not limited to,Anti-Invasive Factor, retinoic acids and paclitaxel (U.S. Pat. No.5,716,981; incorporated herein by reference); AGM-1470 (Ingber et al.,1990; incorporated herein by reference); shark cartilage extract (U.S.Pat. No. 5,618,925; incorporated herein by reference); anionic polyamideor polyurea oligomers (U.S. Pat. No. 5,593,664; incorporated herein byreference); oxindole derivatives (U.S. Pat. No. 5,576,330; incorporatedherein by reference); estradiol derivatives (U.S. Pat. No. 5,504,074;incorporated herein by reference); and thiazolopyrimidine derivatives(U.S. Pat. No. 5,599,813; incorporated herein by reference) are alsocontemplated for use as anti-angiogenic compositions for the combineduses of the present invention.

D. Apoptosis-Inducing Agents

The agents of the present invention that bind copper and form atripartite agent-copper-protein complex may also be combined withtreatment methods that induce apoptosis in any cells within the tumor,including tumor cells and tumor vascular endothelial cells. Althoughmany anti-cancer agents may have, as part of their mechanism of action,an apoptosis-inducing effect, certain agents have been discovered,designed or selected with this as a primary mechanism, as describedbelow.

A number of oncogenes have been described that inhibit apoptosis, orprogrammed cell death. Exemplary oncogenes in this category include, butare not limited to, bcr-abl , bcl-2 (distinct from bcl-1, cyclin D1 ;GenBank accession numbers M14745, X06487; U.S. Pat. Nos. 5,650,491; and5,539,094; each incorporated herein by reference) and family membersincluding Bcl-x1, Mcl-1, Bak, A1, A20. Overexpression of bcl-2 was firstdiscovered in T cell lymphomas. bcl-2 functions as an oncogene bybinding and inactivating Bax, a protein in the apoptotic pathway.Inhibition of bcl-2 function prevents inactivation of Bax, and allowsthe apoptotic pathway to proceed. Thus, inhibition of this class ofoncogenes, e.g., using antisense nucleotide sequences, is contemplatedfor use in the present invention in aspects wherein enhancement ofapoptosis is desired (U.S. Pat. Nos. 5,650,491; 5,539,094; and5,583,034; each incorporated herein by reference).

Many forms of cancer have reports of mutations in tumor suppressorgenes, such as p53. Inactivation of p53 results in a failure to promoteapoptosis. With this failure, cancer cells progress in tumorigenesis,rather than become destined for cell death. Thus, provision of tumorsuppressors are also contemplated for use in the present invention tostimulate cell death. Exemplary tumor suppressors include, but are notlimited to, p53, Retinoblastoma gene (Rb), Wilm's tumor (WT1), baxalpha, interleukin-1b-converting enzyme and family, MEN-1 gene,neurofibromatosis, type 1 (NF1), cdk inhibitor p16, colorectal cancergene (DCC), familial adenomatosis polyposis gene (FAP), multiple tumorsuppressor gene (MTS-1), BRCA1 and BRCA2.

Preferred for use are the p53 (U.S. Pat. Nos. 5,747,469; 5,677,178; and5,756,455; each incorporated herein by reference), Retinoblastoma, BRCA1(U.S. Pat. Nos. 5,750,400; 5,654,155; 5,710,001; 5,756,294; 5,709,999;5,693,473; 5,753,441; 5,622,829; and 5,747,282; each incorporated hereinby reference), MEN-1 (GenBank accession number U93236) and adenovirusE1A (U.S. Pat. No. 5,776,743; incorporated herein by reference) genes.

Other compositions that may be used include genes encoding the tumornecrosis factor related apoptosis inducing ligand termed TRAIL, and theTRAIL polypeptide (U.S. Pat. No. 5,763,223; incorporated herein byreference); the 24 kDa apoptosis-associated protease of U.S. Pat. No.5,605,826 (incorporated herein by reference); Fas-associated factor 1,FAF1 (U.S. Pat. No. 5,750,653; incorporated herein by reference). Alsocontemplated for use in these aspects of the present invention is theprovision of interleukin-1β-converting enzyme and family members, whichare also reported to stimulate apoptosis.

Compounds such as carbostyril derivatives (U.S. Pat. Nos. 5,672,603; and5,464,833; each incorporated herein by reference); branched apogenicpeptides (U.S. Pat. No. 5,591,717; incorporated herein by reference);phosphotyrosine inhibitors and non-hydrolyzable phosphotyrosine analogs(U.S. Pat. Nos. 5,565,491; and 5,693,627; each incorporated herein byreference); agonists of RXR retinoid receptors (U.S. Pat. No. 5,399,586;incorporated herein by reference); and even antioxidants (U.S. Pat. No.5,571,523; incorporated herein by reference) may also be used. Tyrosinekinase inhibitors, such as genistein, may also be linked to ligands thattarget a cell surface receptor (U.S. Pat. No. 5,587,459; incorporatedherein by reference).

IV. Pharmaceutical Compositions and Kits

Pharmaceutical compositions of the present invention will generallycomprise an effective amount of an agent that binds copper and forms atripartite agent-copper-protein complex, such as tetrathiomolybdate,dissolved or dispersed in a pharmaceutically acceptable carrier oraqueous medium.

The phrases “pharmaceutically or pharmacologically acceptable” refer tomolecular entities and compositions that do not produce an adverse,allergic or other untoward reaction when administered to an animal, or ahuman, as appropriate. As used herein, “pharmaceutically acceptablecarrier” includes any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents and the like. The use of such media and agents for pharmaceuticalactive substances is well known in the art. Except insofar as anyconventional media or agent is incompatible with the active ingredient,its use in the therapeutic compositions is contemplated. Supplementaryactive ingredients can also be incorporated into the compositions.

A. Parenteral Formulations

The agents of the present invention that bind copper and form atripartite agent-copper-protein complex, such as tetrathiomolybdate,will often be formulated for parenteral administration, e.g., formulatedfor injection via the intravenous, intramuscular, sub-cutaneous or othersuch routes, including direct instillation into a tumor or disease site.The preparation of an aqueous composition that contains one or moreagents that bind copper and form a tripartite agent-copper-proteincomplex as an active ingredient will be known to those of skill in theart in light of the present disclosure. Typically, such compositions canbe prepared as injectables, either as liquid solutions or suspensions;solid forms suitable for using to prepare solutions or suspensions uponthe addition of a liquid prior to injection can also be prepared; andthe preparations can also be emulsified.

Solutions of the active compounds as free base or pharmacologicallyacceptable salts can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions; formulations including sesame oil,peanut oil or aqueous propylene glycol; and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi.

Compositions comprising the agents of the present invention that bindcopper and form a tripartite agent-copper-protein complex, such astetrathiomolybdate, can be formulated into a composition in a neutral orsalt form. Pharmaceutically acceptable salts, include the acid additionsalts (formed with the free amino groups of the protein) and which areformed with inorganic acids such as, for example, hydrochloric orphosphoric acids, or such organic acids as acetic, oxalic, tartaric,mandelic, and the like. Salts formed with the free carboxyl groups canalso be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, histidine, procaine and thelike.

The carrier can also be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. Formulations are easily administered in a variety of dosageforms, such as the type of injectable solutions described above, butdrug release capsules and the like can also be employed.

Suitable pharmaceutical compositions in accordance with the inventionwill generally include an amount of one or more of the agents of thepresent invention that bind copper and form a tripartiteagent-copper-protein complex, such as tetrathiomolybdate, admixed withan acceptable pharmaceutical diluent or excipient, such as a sterileaqueous solution, to give a range of final concentrations, depending onthe intended use. The techniques of preparation are generally well knownin the art as exemplified by Remington's Pharmaceutical Sciences, 16thEd. Mack Publishing Company, 1980, incorporated herein by reference. Itshould be appreciated that endotoxin contamination should be keptminimally at a safe level, for example, less that 0.5 ng/mg protein.Moreover, for human administration, preparations should meet sterility,pyrogenicity, general safety and purity standards as required by FDAOffice of Biological Standards.

The therapeutically effective doses are readily determinable using ananimal model, as shown in the studies detailed herein. Experimentalanimals bearing solid tumors are frequently used to optimize appropriatetherapeutic doses prior to translating to a clinical environment. Suchmodels are known to be very reliable in predicting effective anti-cancerstrategies. For example, mice bearing solid tumors, such as used inExample 2, are widely used in pre-clinical testing. The inventors haveused such art-accepted mouse models to determine working ranges ofagents such as tetrathiomolybdate that give beneficial anti-tumoreffects with minimal toxicity.

In addition to the compounds formulated for parenteral administration,such as intravenous or intramuscular injection, other pharmaceuticallyacceptable forms are also contemplated, e.g., tablets or other solidsfor oral administration, time release capsules, liposomal forms and thelike. Other pharmaceutical formulations may also be used, dependent onthe condition to be treated. For example, topical formulations may beappropriate for treating pathological conditions such as dermatitis andpsoriasis; and ophthalmic formulations may be appropriate for conditionssuch as diabetic retinopathy. Of course, methods for the determinationof optimal dosages for conditions such as these would be evident tothose of skill in the art in light of the dosage optimizationmethodology disclosed in the instant specification, and the knowledge ofthe skilled artisan.

As described in detail herein, it is contemplated that certain benefitswill result from the manipulation of the agents of the present inventionthat bind copper and form a tripartite agent-copper-protein complex,such as tetrathiomolybdate, to provide them with a longer in vivohalf-life. Slow release formulations are generally designed to give aconstant drug level over an extended period. Increasing the half-life ofa drug, such as agents of the present invention that bind copper andform a tripartite agent-copper-protein complex, for exampletetrathiomolybdate, is intended to result in high plasma levels uponadministration, which levels are maintained for a longer time, but whichlevels generally decay depending on the pharmacokinetics of theconstruct. Although currently not preferred, slow release formulationsof the instant compositions and combinations thereof are by no meansexcluded from use in the present invention.

B. Therapeutic Kits

The present invention also provides therapeutic kits comprising theagents of the present invention that bind copper and form a tripartiteagent-copper-protein complex, such as tetrathiomolybdate, describedherein. Such kits will generally contain, in suitable container means, apharmaceutically acceptable formulation of at least one agent that bindscopper and forms a tripartite agent-copper-protein complex, such astetrathiomolybdate, in accordance with the invention. The kits may alsocontain other pharmaceutically acceptable formulations, such as any oneor more of a range of chemotherapeutic drugs.

The kits may have a single container means that contains the agent thatbinds copper and forms a tripartite agent-copper-protein complex, suchas tetrathiomolybdate, with or without any additional components, orthey may have distinct container means for each desired agent. Certainpreferred kits of the present invention include an agent that bindscopper and forms a tripartite agent-copper-protein complex, such astetrathiomolybdate, packaged in a kit for use in combination with theco-administration of a second anti-cancer agent, such as achemotherapeutic agent, a radiotherapeutic agent, a distinct copperchelating agent, an anti-angiogenic agent or an apoptosis-inducingagent. In such kits, the components may be pre-complexed, either in amolar equivalent combination, or with one component in excess of theother; or each of the components of the kit may be maintained separatelywithin distinct containers prior to administration to a patient.

When the components of the kit are provided in one or more liquidsolutions, the liquid solution is an aqueous solution, with a sterileaqueous solution being particularly preferred. However, the componentsof the kit may be provided as dried powder(s). When reagents orcomponents are provided as a dry powder, the powder can be reconstitutedby the addition of a suitable solvent. It is envisioned that the solventmay also be provided in another container means. One of the componentsof the kit may be provided in capsules for oral administration.

The container means of the kit will generally include at least one vial,test tube, flask, bottle, syringe or other container means, into whichthe agent that binds copper and forms a tripartite agent-copper-proteincomplex, such as tetrathiomolybdate, and any other desired agent, may beplaced and, preferably, suitably aliquoted. Where additional componentsare included, the kit will also generally contain a second vial or othercontainer into which these are placed, enabling the administration ofseparated designed doses. The kits may also comprise a second/thirdcontainer means for containing a sterile, pharmaceutically acceptablebuffer or other diluent.

The kits may also contain a means by which to administer the agent thatbinds copper and forms a tripartite agent-copper-protein complex, suchas tetrathiomolybdate, to an animal or patient, e.g., one or moreneedles or syringes, or even an eye dropper, pipette, or other such likeapparatus, from which the formulation may be injected into the animal orapplied to a diseased area of the body. The kits of the presentinvention will also typically include a means for containing the vials,or such like, and other component, in close confinement for commercialsale, such as, e.g., injection or blow-molded plastic containers intowhich the desired vials and other apparatus are placed and retained.

V. Cancer and Treatment

The compositions and methods provided by this invention are broadlyapplicable to the treatment of any malignant tumor having a vascularcomponent. Typical vascularized tumors are the solid tumors,particularly carcinomas and sarcomas, which require a vascular componentfor the provision of oxygen and nutrients. Hematologic malignancies alsoappear to require angiogenesis for progression, and thus are alsopotentially amenable to treatment with the instant copper loweringagents. Exemplary solid tumors that may be treated using the inventioninclude, but are not limited to, primary carcinomas of the lung, breast,ovary, stomach, pancreas, larynx, esophagus, testes, liver, parotid,biliary tract, colon, rectum, cervix, uterus, endometrium, kidney,bladder, prostate, thyroid, squamous cell carcinomas, adenocarcinomas,small cell carcinomas, melanomas, gliomas, neuroblastomas, sarcomas,such as angiosarcomas and chondrosarcomas, and the like. Metastatictumors may also be treated using the methods and compositions of thepresent invention.

The present invention is contemplated for use in the treatment of anypatient that presents with a solid tumor. However, in that the presentinvention is particularly successful in the treatment of solid tumors ofmoderate or large sizes, patients in these categories are likely toreceive more significant benefits from treatments in accordance with themethods and compositions provided herein. In general, the invention canbe used to treat tumors of about 0.3-0.5 cm and upwards, although tumorsup to and including the largest tumors found in humans may also betreated.

In certain aspects of the present invention, the agents such astetrathiomolybdate are intended as a preventative or prophylactictreatment and as a maintenance agent. There are many reasons underlyingthis aspect of the breadth of the invention. For example, a patientpresenting with a primary tumor of moderate size or above may also havevarious other metastatic tumors that are considered to be small-sized oreven in the earlier stages of metastatic tumor seeding. Given that TMand combinations of the invention are generally administered orally orinto the systemic circulation of a patient, they will naturally haveeffects on the secondary, smaller and metastatic tumors, although thismay not be the primary intent of the treatment. Furthermore, even insituations where the tumor mass as a whole is a single small tumor,certain beneficial anti-tumor effects will result from the use of thepresent treatments.

The guidance provided herein regarding the most suitable patients foruse in connection with the present invention is intended as teachingthat certain patient's profiles may assist with the selection ofpatients that may be treated by the present invention, or that may,perhaps, be better treated using other anti-cancer treatment strategies.Nonetheless, the fact that a preferred or otherwise more effectivetreatment is perceived to exist in connection with a certain category ofpatients, does not in any way negate the basic utility of the presentinvention in connection with the treatments of all patients having avascularized tumor. A further consideration is the fact that the initialassault on a tumor, as provided by the therapy of the present invention,may be small in any measurable and immediate effects, but may sensitizeor potentiate the tumor to further therapeutic treatments such that thesubsequent treatment results in an overall synergistic effect or evenleads to total remission or cure.

It is not believed that any particular type of tumor should be excludedfrom treatments using the present invention. It will be understood thatthe present methodology is widely or entirely applicable to thetreatment of all solid tumors, irrespective of the particular phenotypeor genotype of the tumor cells themselves. However, the type of tumorcells may be relevant to the use of the invention in combination withsecondary therapeutic agents.

Those of ordinary skill in the art will understand that certain types oftumors may be more amenable to the induction of tumor stasis,regression, and even necrosis using the present invention. The phenomenais observed in experimental animals, and may occur in human treatments.Such considerations will be taken into account in conducting both thepre-clinical studies in experimental animals and in optimizing the dosesfor use in treating any particular patient or group of patients.

As detailed herein, there are realistic objectives that may be used as aguideline in connection with pre-clinical testing before proceeding toclinical treatment. However, this is more a matter of cost-effectivenessthan overall usefulness, and is a mechanism for selecting the mostadvantageous compounds and doses. In regard to their basic utility, anyconstruct or combination thereof that results in any consistentanti-tumor effects will still define a useful invention. It will also beunderstood that even in such circumstances where the anti-tumor effectsof the instant compositions and combinations thereof are towards the lowend of the range, it may be that this therapy is still equally or evenmore effective than all other known therapies in the context of theparticular tumor targets. It is unfortunately evident to a clinicianthat certain tumors cannot be effectively treated in the intermediate orlong term, but that does not negate the usefulness of the presenttherapy, particularly where it is about as effective as the otherstrategies generally proposed, or it may be effective after all otherconventional strategies have failed. It is not predicted that resistanceto this therapy can develop.

In designing appropriate doses of the agents that bind copper and form atripartite agent-copper-protein complex, and combinations therewith, onemay readily extrapolate from the animal studies described herein inorder to arrive at appropriate doses for clinical administration. Toachieve this conversion, one would account for the mass of the agentsadministered per unit mass of the experimental animal, and yet accountfor the differences in the body surface area between the experimentalanimal and the human patient. All such calculations are well known androutine to those of ordinary skill in the art. Accordingly, using theinformation provided herein, the inventors contemplate that useful dailydoses of the agents that bind copper and form a tripartiteagent-copper-protein complex, such as tetrathiomolybdate, for use inhuman administration would be between about 20 milligrams and about 200milligrams per patient per day. Notwithstanding this stated range, itwill be understood that, given the parameters and detailed guidancepresented above, further variations in the active or optimal rangeswould still be encompassed within the present invention.

The daily doses contemplated will therefore generally be between about20 mg and about 180 milligrams; between about 130 mg and about 200milligrams; between 25 and about 160 milligrams; between 50 and about150 milligrams; between about 150 mg and about 180 milligrams; betweenabout 30 and about 125 milligrams; between about 40 and about 100milligrams; between about 35 and about 80 milligrams; between about 140mg and about 190 milligrams; between about 20 and about 65 milligrams;between about 125 mg and about 195 milligrams; between about 30 andabout 50 milligrams; between about 150 mg and about 200 milligrams; orin any particular range using any of the foregoing recited exemplarydoses or any value intermediate between the particular stated ranges.

Although doses in and around about 60-120 mg are currently preferred incertain embodiments of the present invention, and doses in and aroundabout 125-200 mg are currently preferred in other embodiments of thepresent invention, it will be understood that lower doses may be moreappropriate in combination with other agents, or under conditions ofmaintenance, and that high doses can still be tolerated, particularlygiven the fact that the agents that bind copper and form a tripartiteagent-copper-protein complex for use in the invention are not themselvescytotoxic and even if certain adverse side effects do occur, this shouldnot necessarily result in toxicity that cannot be counteracted by normalhomeostatic mechanisms, which is believed to lessen the chances ofsignificant toxicity to healthy tissues.

In certain preferred embodiments of the present invention, daily loadingdosages of between about 130 mg or about 150 mg or so to about 180 mg orabout 200 mg or so are administered to patients for about 2 weeks,followed by daily maintenance dosages of between about 30 mg or about 40mg or so and about 60 mg or about 70 mg or so, or any valuesintermediate between the particular stated ranges. Thus, in particularaspects of the invention, loading dosages of greater than about 125 mg,greater than about 130 mg, greater than about 140 mg, greater than about150 mg, greater than about 155 mg, greater than about 160 mg, greaterthan about 170 mg, greater than about 175 mg, greater than about 180 mg,greater than about 190 mg, or greater than about 200 mg or so up to themaximum dosages described herein are contemplated by the inventors asexemplary daily loading dosages for about 1 week, about 2 weeks, about 3weeks or about 4 weeks or so, followed by daily maintenance dosages ofabout 20 mg, about 25 mg, about 35 mg, about 40 mg, about 50 mg, about55 mg, about 65 mg, about 75 mg, about 80 mg or about 90 mg or so.

The intention of the therapeutic regimens of the present invention isgenerally to produce the maximum anti-tumor effects whilst still keepingthe dose below the levels associated with unacceptable toxicity. Inaddition to varying the dose itself, the administration regimen can alsobe adapted to optimize the treatment strategy. A currently preferredtreatment strategy is to administer between about 20 milligrams andabout 200 milligrams of the agents that bind copper and form atripartite agent-copper-protein complex or combination thereof about 3,about 4, about 5 to about 6 or more times a day, approximately half ofthe time with meals, and approximately half of the time between meals.In administering the particular doses themselves, one would preferablyprovide a pharmaceutically acceptable composition to the patientsystemically. Oral administration is generally preferred.

VI. Other Diseases Characterized by Aberrant Angiogenesis

In addition to the prevention or treatment of cancer and solid tumors,the thiomolybdate compositions disclosed herein can also be used inpreventing or treating other diseases associated with aberrantvascularization, including, but not limited to, arthritis, diabetes,arteriosclerosis, arteriovenous malformations, corneal graftneovascularization, delayed wound healing, diabetic retinopathy, agerelated macular degeneration, granulations, burns, hemophilic joints,rheumatoid arthritis, hypertrophic scars, neovascular glaucoma, nonunionfractures, Osier-Weber Syndrome, psoriasis, pyogenic granuloma,retrolental fibroplasia, pterygium, scleroderma, trachoma, vascularadhesions, ocular neovascularization, parasitic diseases, hypertrophyfollowing surgery, and inhibition of hair growth.

Macular degeneration is the common name for the age-related diseasewhere macular retinal pigment epithelium cells function less well thannormal. As a result, waste removal and nutrition of the cones suffers,causing central vision loss. Macular degeneration can be furtherclassified into two varieties: a “dry type” and a “wet type”. Dry typemacular degeneration occurs when the outer segments of the light sensingcones, which are continuously being shed, are unable to be digested bythe pigment epithelium layer of the macula. Consequently the pigmentepithelium layer swells and eventually dies after accumulating too muchundigested material from the cones. Yellowish deposits of this wastematerial gradually develop under the retina between the choroid andpigment epithelium. In this “dry type” macular degeneration, the visionloss is characterized by gradual blurring or partial obscuration ofcentral vision as a result of parts of the macula having begun to die,creating areas where the cones are no longer functional. Clinically, theperson suffering from this type of the disease may experience relativelymild central visual distortion with straight lines appearing bent orwavy.

In the second or “wet” type of this disorder, more severe and suddenvision loss may occur. This occurs when abnormal new blood vessels or“neovascular membranes” grow from the choroid through the damagedpigment epithelium and under the macula. These neovascular membranes arefragile and are prone to hemorrhage, which results in severe distortionof the macular tissue. As a result, the light sensing cells (cones)become separated from their source of nutrients and suffer furtherdamage due to scarring as the hemorrhage occurs over time. With thistype of disorder, dark or “missing” spots in the central vision mayoccur rapidly and with little warning due to these hemorrhagic changes.Fortunately, intervention with laser therapy early in this process mayprevent additional vision loss.

Age-related macular degeneration (AMD) is the leading cause of visualloss among adults aged 65 years or older in Western countries. Althoughneovascular AMD accounts for only 10% of all cases, it is responsiblefor 80% to 90% of legal blindness due to this disease and is the mostcommon cause of choroidal neovascularization (CNV) in this agepopulation. The pathological changes leading to CNV involve the complexof tissues in the choriocapilaris, Bruch's membrane, and the retinalpigment epithelium (RPE) with secondary involvement of the neurosensoryretina. Essentially anything that alters the retinal pigment epitheliumand Bruch's membrane can cause CNV.

A variety of conditions other than AMD have been associated with CNV,including ocular histoplasmosis syndrome (POHS), pathologic myopia,angioid streaks, and idiopathic causes. Most histopathological studieshave been performed in eyes with AMD. The histopathological featurecommon to many eyes that develop CNV is a break in Bruch's membrane. Thecapillary-like neovascularization originates from choroidal vessels andextends through these breaks. Age-related macular degeneration accountsfor the largest group of patients with CNV. Most symptomatic CNV's aresubfoveal and demonstrate an extremely poor natural history. Subfovealneovascularization is defined as lesions lying under the geometriccenter of the foveal avascular zone (FAZ). Of untreated eyes followedfor 2 years in a Macular Photocoagulation Study (MPS), only 5% had afinal visual acuity better than 20/100, whereas 88% had a final visualacuity of 20/200 or worse.

Laser photocoagulation has been the mainstay of therapy for choroidalneovascularization. Through a series of well-executed randomized,prospective clinical trials, the MPS established the superiority ofphotocoagulation over observation for CNV in a variety of settings.Specifically, photocoagulation treatment of extrafoveal and juxtafovealneovascular membranes in AMD and other disorders was found to bebeneficial compared to the no treatment group. However, in order totreat the entire area of CNV, the ophthalmologist has to be able toidentify the boundaries of the choroidal neovascular membrane.Therefore, treatment is indicated only when the boundaries of the CNVare well demarcated. Unfortunately, occult or ill-defined new vesselsare the most common pattern at presentation for exudative macularlesions in AMD. In one study, visible or classic neovascular membranesinvolved only 23% of eyes referred for treatment. The MPS recentlyreported results of photocoagulation for subfoveal neovascular lesionsin AMD showed benefit of laser treatment, but the difference between thetreatment and observation groups was small and was seen only after twoand five years. Also, as the laser energy destroys both the retina andsubretinal membrane, there was a precipitous drop in visual acuityassociated with treatment. These results underline both the poor naturalhistory of the condition and the limitations of photocoagulation as atreatment modality.

Other diseases associated with corneal neovascularization include, butare not limited to, epidemic keratoconjunctivitis, Vitamin A deficiency,contact lens overwear, atopic keratitis, superior limbic keratitis,pterygium keratitis sicca, sjogrens, acne rosacea, phylectenulosis,syphilis, Mycobacteria infections, lipid degeneration, chemical burns,bacterial ulcers, fungal ulcers, Herpes simplex infections, Herpeszoster infections, protozoan infections, Kaposi sarcoma, Mooren ulcer,Terrien's marginal degeneration, marginal keratolysis, rheumatoidarthritis, systemic lupus, polyarteritis, trauma, Wegeners sarcoidosis,Scleritis, Steven's Johnson disease, periphigoid radial keratotomy, andcorneal graph rejection.

Diseases associated with retinal/choroidal neovascularization include,but are not limited to, diabetic retinopathy, macular degeneration,sickle cell anemia, sarcoid, syphilis, pseudoxanthoma elasticum, Pagetsdisease, vein occlusion, artery occlusion, carotid obstructive disease,chronic uveitis/vitritis, mycobacterial infections, Lyme's disease,systemic lupus erythematosis, retinopathy of prematurity, Eales disease,Bechets disease, infections causing a retinitis or choroiditis, presumedocular histoplasmosis, Bests disease, myopia, optic pits, Stargartsdisease, pars planitis, chronic retinal detachment, hyperviscositysyndromes, toxoplasmosis, trauma and post-laser complications. Otherdiseases include, but are not limited to, diseases associated withrubeosis (neovasculariation of the angle) and diseases caused by theabnormal proliferation of fibrovascular or fibrous tissue including allforms of proliferative vitreoretinopathy.

Another disease in which angiogenesis is believed to be involved isrheumatoid arthritis. Rheumatoid arthritis is characterized by diffuseand nodular mononuclear cell infiltration and massive hyperplasia of thestromal connective tissues, comprised of fibroblast-like cells and newblood vessels. The blood vessels in the synovial lining of the jointsundergo angiogenesis. In addition to forming new vascular networks, theendothelial cells release factors and reactive oxygen species that leadto pannus growth and cartilage destruction. The factors involved inangiogenesis may actively contribute to, and help maintain, thechronically inflamed state of rheumatoid arthritis.

Factors associated with angiogenesis may also have a role inosteoarthritis. The activation of the chondrocytes by angiogenic-relatedfactors contributes to the destruction of the joint. At a later stage,the angiogenic factors would promote new bone formation. Chronicinflammation may also involve pathological angiogenesis. Such diseasestates as ulcerative colitis and Crohn's disease show histologicalchanges with the ingrowth of new blood vessels into the inflamedtissues. Bartonellosis, a bacterial infection found in South America,can result in a chronic stage that is characterized by proliferation ofvascular endothelial cells. Another pathological role associated withangiogenesis is found in atherosclerosis. The plaques formed within thelumen of blood vessels have been shown to have angiogenic stimulatoryactivity.

VII. Wilson's Disease

A. Background

Wilson's disease is an autosomal recessive disorder of coppermetabolism. In this disorder, the excretion of copper into the bileappears to be defective, and there is reduced hepatic incorporation ofcopper into ceruloplasmin, leading to an accumulation of copper inplasma and most body tissues. Wilson's disease usually leads to hepaticand/or neurologic dysfunction.

The therapy of Wilson's disease can be divided into two broad categories(Brewer and Yuzbasiyan-Gurkan, 1992a). These are initial therapy inacutely ill patients, and maintenance therapy. Initial therapy is thatperiod of time during which a newly presenting patient is stillsuffering from acute copper toxicity, generally the first few weeks tomonths of therapy. Maintenance therapy is essentially the rest of thepatient's life, or that period of time after the copper levels have beenbrought down to a subtoxic threshold, and the patient is on therapysimply to prevent the recurrence of copper accumulation and coppertoxicity.

For the maintenance therapy of Wilson's disease three drugs arecurrently available. These include the oldest available drug,penicillamine (Walshe, 1956), a drug called trien or trientine which wasdeveloped for patients who are intolerant of penicillamine (Walshe,1982), and zinc acetate (Brewer and Yuzbasiyan-Gurkan, 1992a, b; Breweret al., 1983; Hill et al., 1987; Hill et al., 1986; Brewer et al.,1987b, c, d; Yuzbasiyan-Gurkan et al., 1989; Brewer et al., 1989; Lee etal., 1989; Brewer et al., 1990; Brewer et al., 1991b; Brewer andYuzbasiyan-Gurkan, 1989; Brewer et al., 1992a, b; Yuzbasiyan-Gurkan etal., 1992; Brewer et al., 1993a, b, c, d; Brewer, 1993; Hoogenraad etal., 1978; Hoogenraad et al., 1979; Hoogenraad et al., 1987). Zincprovides an effective maintenance therapy with a very low level oftoxicity.

About ⅔ of patients who present with Wilson's disease present withsymptoms referable to the brain (Brewer et al., 1992a; Scheinberg andSternlieb, 1984; Danks, 1989). These can be neurologic symptoms orsymptoms of psychiatric nature in the beginning, with neurologicsymptoms later. Therapy for these patients is not nearly asstraightforward as it is for maintenance phase patients. The inventorshave found that approximately 50% of these patients who are treated withpenicillamine become worse rather than better (Brewer et al., 1987a).Half of these patients who worsen, or about 25% of the original sample,never recover to their pre-penicillamine baseline. In other words,penicillamine has induced additional irreversible damage.

The mechanisms of this worsening are not known with certainty althoughit is likely that the mobilization of hepatic copper by the drug furtherelevates brain copper. The inventors have shown that this can occur in arat model. Regardless of the mechanism, neurologically-presentingpatients very often end up much worse off after being treated initiallywith penicillamine. In fact, even presymptomatic patients can developneurologic disease after being initiated on penicillamine (Glass et al.,1990; Brewer et al., 1994a). It is not known whether trientine exhibitsthe phenomenon of neurological worsening when used as initial therapy,because it has not been used very much in this kind of situation. Itwould not be surprising if it exhibited this problem to some degreebecause of it's similar mechanism of action to that of penicillamine,but it might be much less, because its effects on copper seem to besomewhat gentler.

Zinc is not an ideal agent for the initial treatment for this type ofpatient. Zinc has a relatively slow onset of action, and produces only amodest negative copper balance. Thus, during the several months requiredfor zinc to bring copper down to a subtoxic threshold, patients may beat risk for further copper toxicity and worsening of their disease.

B. Results of TM Therapy

The inventors have carried out an open label study of the use of TM forinitial treatment of neurologically presenting Wilson's disease patientsfor the past several years. The inventors have developed both aspectrophotometric and bioassay for the activity of the drug, toevaluate stability, and assure potency of the drug being administered(Brewer et al., 1991a; Brewer et al., 1994b). The drug slowly losespotency when exposed to air. Oxygen molecules exchange with the sulfurmolecules, rendering the drug inactive.

The results in the first patient studied can be used to illustrateseveral points. For the first seven days, the patient received TM onlywith meals (tid with meals). This produced the immediate negative copperbalance one would expect from the first mechanism of action (blockade ofcopper absorption when given with meals). After the first seven days, TMwas given between meals as well (tid with meals, and tid between meals).This led to the immediate rise in plasma copper expected from absorptionof TM into the blood, and formation of a complex of copper, TM, andalbumin. The copper complexed with TM and albumin is unavailable forcellular uptake, and this copper is therefore non-toxic (Gooneratne etal., 1981b). There is a 1:1 stoichometric relationship betweenmolybdenum and copper in this complex. Knowing the molybdenum level inthe blood, and the ceruloplasmin level (ceruloplasmin also containscopper that is non-toxic), one can calculate how much of the plasmacopper is not bound to one or the other. This so-called “free copper”(non-ceruloplasmin plasma copper) is the potentially toxic copper. Whenreduced to zero, the plasma copper-molybdenum “gap” is closed. This took16 days in the first patient (9 days after adding the between mealdoses). Since the brain (and the other organ) free copper is inequilibrium with the blood, bringing the blood free copper down to a lowlevel begins the process of lowering the brain level of free (toxic)copper.

The inventors have now treated a total of 51 Wilson's disease patientswith TM, all of whom presented with neurological or psychiatric disease,in an open label study. These patients were all diagnosed by standardcriteria. These patients had a diagnostically elevated hepatic or urinecopper, usually both. Some of them were treated briefly with otheragents prior to this trial. Two patients had psychiatric but notneurological symptoms.

With three exceptions in the earliest part of the study, all patientsreceived a dose of 20 mg tid with meals, or qid with three meals and asnack. Thus, the only difference between a patient receiving 120 mg and140 mg total dose is that the former was receiving 20 mg tid, or 60 mg,with meals, and the latter was receiving 20 mg qid, or 80 mg with mealsplus a snack. The rest of the total daily dose was divided up into threeequal doses and given between meals.

The total daily dose was varied considerably among the patients, from ahigh of 410 mg to a low of 120 mg. In the end, the inventors coulddiscern no dose-related correlation with copper variables, nor withfunctional variables measured either during the study or at the one andtwo year time point.

Zinc administration was also used in these patients. The starting timeof zinc administration was varied widely and did not correlate withcopper variables, outcome variables or toxicity. Early zinc therapyshould theoretically help preserve liver function. In these patients,liver function returned to normal by year 1, but since these tests don'tmeasure the extent of tissue preservation, it seems likely that zinc wassomewhat beneficial.

Measuring trichloracetic acid (TCA) soluble copper of the plasma issomewhat useful in assessing the impact of TM therapy on coppermetabolism in Wilson's disease. Generally, a high proportion of plasmacopper in these patients is TCA soluble (it averaged 56% inpatients—which is 27 μg/dl). All of the non-ceruloplasmin plasma copperis TCA soluble, and a somewhat variable portion of the ceruloplasmincopper is also TCA soluble. Because the ceruloplasmin levels are usuallyrather low in Wilson's disease, most of the plasma copper is TCAsoluble. The copper in the TM/albumin/copper complex in the blood is TCAinsoluble. Thus, as therapy proceeds, the fraction of the plasma copperwhich is TCA soluble becomes smaller. During the late stages of TMtherapy, the TCA soluble fraction of plasma copper of the patientsaveraged 15 μg/dl, a significant reduction from the starting value of27. The TCA solubility fraction cannot be used as an absolute endpoint,for example attempting to reduce it to zero, because a small andsomewhat variable soluble fraction is usually present due to plasmaceruloplasmin. However, the significant mean reduction from 27 to 15μg/dl illustrates the beneficial effect TM therapy has on the status ofthe potentially toxic plasma copper in these patients. Further evidenceof the desirable impact of TM therapy on copper metabolism is shown byreduction of mean urine copper values during the latter part of TMtherapy, compared to baseline values.

TM impacts quickly and favorably on copper metabolism, reducing thepotentially toxic copper of the blood and theoretically, the rest of thebody. The primary clinical objective is to gain control over coppertoxicity while not allowing clinical worsening. In other words, theprime objective is to protect all neurological function that is presentat the time therapy is started. This was evaluated weekly byquantitative neurological and speech exams. Methodology and theneurology rating scale system have been published (Young et al., 1986).During the weeks of TM administration, during which copper metabolism isbeing controlled, neurological function as evaluated by quantitativeneurological exam, is protected. Only two patients (4% of the sample)showed a change of more than 5 units, the criterion for significantworsening.

During the following years, while the patients are on maintenancetherapy, the brain damage previously induced by copper is at leastpartially repaired. This is exemplified by the partial recovery inneurological scores seen at yearly time-points in follow-up. It is clearthat with the initial TM approach, long term recovery is excellent, mostpatients showing substantial neurological recovery. These excellentresults are to be contrasted with penicillamine therapy. As pointed outearlier, about 50% of patients initially deteriorate on penicillamine,and that half of these, or 25% of the original sample, never recover totheir pre-penicillamine baseline.

The results during the initial 8 weeks of TM therapy on quantitativespeech exams are performed as described (Brewer et al. 1996). During theweeks of TM administration, during which copper metabolism is beingcontrolled, neurological function as measured by quantitative speechexams is being controlled. No patient shows significant (more than 2.0units) reduction in scores. During the following years, while thepatients are on maintenance therapy, the brain damage previously inducedby copper is partially repaired. This is exemplified by the partialrecovery in speech scores over years of follow-up. Long term recovery isexcellent. No patient shows significantly (more than 2.0 units) lesslong term function than at the time of initiation of therapy, and mostshow marked improvement.

Two undesirable effects from TM therapy were observed in these patients.One is a reversible anemia/bone marrow depression, which was exhibitedby seven patients. The fall in hemoglobin in all of these patients wassignificant, averaging 3.4 g %. Three of the patients showed a reductionin platelet count and four of the patients showed a reduction in whiteblood cell count that may have been significant. TM was stopped in allseven cases. Except for two of the patients, stoppage was late in the 56day course of TM.

At the time of the anemia, these patients all had zero non-ceruloplasminplasma copper and an extremely low TCA soluble copper. The latteraveraged 2.7 in these patients, and the average value for this variablein the entire group of patients was 27 at the beginning and 15 at theheight of therapy. The cause of the anemia/bone marrow depression wasconcluded to be bone marrow depletion of copper. Since copper isrequired for heme synthesis and other steps in cell proliferation, itcould be expected that anemia and bone marrow effect would be the firstsigns of copper depletion. This result from copper depletion is awell-known phenomenon.

Thus, this undesirable response to TM is not a side effect but is,rather, due to overtreatment. It is perhaps surprising that it ispossible to produce even localized bone marrow copper depletion withinsuch a short period of time in Wilson's disease, a disease in which thebody is overloaded with copper. This response to TM is unique. None ofthe other anti-copper drugs are able to produce this effect in earlytherapy. Thus, this speaks to the potency of TM and the rapidity withwhich it can control copper levels. Its also likely that the bone marrowis especially dependent on plasma copper, and that it is the first poolthat it is reduced to very low levels. At a dose of 180 mg/day or over,overtreatment occurred in 6 of 37 patients. At a dose of 150 or lower,only 1 of 13 patients exhibited overtreatment, and that was very late(53 days in the 56 day program).

The second undesirable effect of TM therapy in these patients is anelevation of transaminase values in four of the patients. The serum ASTand ALT values were elevated. TM therapy was discontinued in one patientbecause of these elevations. During these elevations, the urine copperincreases, contrary to the general trend in other patients, where it isdecreasing. These data support the concept that a hepatitis isoccurring, with release of copper from damaged hepatocytes. It is notclear why this hepatitis is occurring. However, untreated Wilson'sdisease patients have a episodic hepatitis as part of their history.Since there is little in the way of observation of untreated patientsafter diagnosis, no good information exists on how often episodes oftransaminase elevations occur as part of the natural history of thedisease.

Alternatively, the TM in some cases may be mobilizing hepatic copper ata faster rate than it can be disposed of, in which case these patientswould be classified as showing a side effect of treatment. Against thisis the observation in copper-poisoned sheep, in which the acutehepatitis, liver necrosis, and hemolytic anemia are rapidly correctedwith high doses of TM. All four of these patients were treated with 150mg TM/day or higher. None of the patients treated with 150 mg or lowerexhibited this response. No other negative effects of TM have beenobserved.

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventors to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

EXAMPLE 1 Effect of TM on Cellular Toxicity

Normal and neoplastic cells in culture derive their nutrients fromtransport and utilization of molecules from the media to the interior ofthe cell. This process is not dependent on blood vessel growth, andtherefore TM should have no effect on cell growth rates and cellviability over a wide range of concentration, until a level of TM whichis toxic to most cells is reached. One mechanism of toxicity is thedepletion of free copper levels below those required for basic cellfunction. For concentrations of TM beyond this toxic level, both normaland neoplastic cells will be unable to survive, due to cellular toxicityof TM.

This was confirmed in a cytotoxicity assay of MML cells (prostatecancer) and breast cancer cells. After plating cells in equal numbers inmedia containing various concentrations of TM ranging from 0.001 μM to 1mM, no toxicity was observed in the range of concentrations used invivo. The fraction of viable cells decreased precipitously from 100% to15% when the concentration of TM increased from 1 μg/ml to 10 μg/ml. TMis therefore not contemplated for use a direct cytotoxic agent, as it isclear that, at the very high doses required for a cytotoxic effect, bothtumor and normal cell death would occur. The serum TM concentrationsneeded to achieve an effective decoppering level are 100-500-fold lowerthan the threshold lethal level for cells. When used at efficaciousdecoppering doses in mice and humans with Wilson's disease, TM has notresulted in any clinically apparent direct cell toxicity.

EXAMPLE 2 Use of TM in Murine Pre-Clinical Anti-Cancer Studies

The inventors reasoned that a greater degree of copper deficiency thanpreviously achieved is necessary to significantly inhibit angiogenesisand arrest tumor growth. This means that, in addition to decreased tumormass, prolonged survival or tumor regression would also be observed. Thestudies described below, which used anti-copper approaches for tumorgrowth inhibition in rodents, did not fully incorporate guidelinesderived from human and animal trace element studies in general, andcopper studies in particular (Dick and Bull, 1945; Miller and Engel,1960; Macilese Ammerman et al., 1969; Mills et al., 1958; Cox et al.,1960; Dick et al., 1975; Mason, 1990; McQuaid and Mason, 1991; Mills etal., 1981a; Mills et al., 1981b; Bremner et al., 1982; Gooneratne etal., 1981a, b; Jacob et al., 1981).

In contrast, the present invention uses TM, the most potent anti-copperagent known. The inventors' extensive experience using zinc as atreatment in sickle cell anemia (where the inventors described the firsthuman cases of zinc induced copper deficiency; Brewer et al., 1983),zinc as an anti-copper treatment in Wilson's disease (Brewer et al.,1989; Brewer, 1995a), and TM for the initial anti-copper treatment inWilson's disease (Brewer et al., 1983; Brewer, 1992; Brewer et al.,1994b; Brewer et al., 1995b) was employed in the development ofalgorithms to achieve effective copper deficiency. As the developers ofTM for clinical use, the inventors have acquired significant experiencewith TM in both animals and humans.

A. Injection of Tumor Cells into C567B1/C6 Mice

Under these guidelines, anti-angiogenesis therapeutic studies wereconducted using TM in a mouse tumor model. This study involvedsubcutaneous or intramuscular injection of tumor cells into young adultC57B1/6J mice. Two tumors were used, a mouse sarcoma, MCA205, and amouse melanoma, B16B16. The MCA 205 tumor cell suspension was injectedsubcutaneously into C57B16 mice. After the tumors were palpable, copperdeficiency was induced by use of TM in half the animals, using Cp levelsto monitor copper status. The other half were sham treated. Tumor growthwas compared between the untreated control group and mice treated withsufficient TM to reduce Cp to about 10% of normal controls.

A significant effect on reducing the growth rate of tumors, and onreducing final tumor size and weight, was obtained with TM. Tumor growthwas slowed by the treatment, although not halted, in the relativelybrief period of observation (16 days). The tumors in TM-treated miceweighed significantly less than the tumors in control mice, normalizedto the total weight of the animal. The results were similar in bothtumor types. Thus TM reduced tumor growth and mass, presumably via itsanti-angiogenesis mechanism, as there are no cytotoxic effect at thedoses employed.

However, this experimental design can be improved further to evaluatethe potential efficacy of TM as an anti-tumor agent for the followingreasons: 1) In the mouse, tumors over 1 cm can become a life-threateningburden, and often ulcerate. However, in both mice and humans, it istumors of this size and larger which are most dependent uponangiogenesis for sustained growth; and 2) The period between detectingthe tumor at 2-3 mm and at 1.5 cm is relatively brief in the mouse,consisting of only a few days. As it takes time to deplete the tumormass of copper (especially since most tumors sequester high levels ofcopper), the tumor may support significant angiogenesis from its ownstores, prior to the achievement of near complete copper depletion.

B. HER2-neu Transgenic Mice

Therefore, an animal protocol was designed to test the effectiveness ofTM in retarding or preventing clinically evident tumors in cancer pronefemale HER2-neu transgenic mice (Guy et al., 1992; Muller et al., 1988).These mice are normal at birth and in infancy, but because of thetransgene, one hundred percent of these mice develop mammary tumorsbetween 4-8 months of age (median 205 days) (Guy et al., 1992). A majoradditional reason for choosing this model is that the natural history ofthese murine mammary tumors is remarkably akin to the clinical behaviorof untreated breast cancer in humans. The HER2-neu mouse tumors developafter a long latency (now known to be likely due to mutagenesis of thetransgene and not to over expression) and they remain primarilylocal-regional until they achieve a large size (often >2.5 cm) beforemetastasizing mostly to lung. The time of onset of tumors and thequality and quantity of tumor vessels between the treatment and controlgroups were compared.

After breeding 3 founder transgenic females with 3 transgenic males, thefemale progeny was segregated into 2 groups: one group had 15 treatmentand the other 22 control animals. The percentage of disease-free mice inthe untreated control group was compared to a group of the sametransgenic mice treated with 0.5-1.0 mg TM by gavage daily, starting at100 days of age. Treatment with TM was initiated approximately 80-100days before the tumors would have become clinically evident, so that thetreated animals are rendered copper deficient throughout the key periodof tumor development when angiogenesis may begin to be required. Thisprevents tumors from sequestering large amounts of copper from whichthey could sustain angiogenesis, even in the face of falling total bodycopper.

With a median follow-up of 260 days, none of the TM-treated micedeveloped clinically overt tumors, whereas 70% of the controls had showntumors in the same follow-up period (FIG. 1). Fifty percent of controlmice had developed clinically apparent tumors by age 218 days (p<0.02).Whereas the controls started exhibiting tumors beginning at age 153days, the TM-treated animals showed no tumors until TM-therapy wasdiscontinued and copper levels were allowed to drift upwards (p<0.0146).

Monitoring of blood copper levels in the treatment group by use of asurrogate indicator, ceruloplasmin (Cp), revealed that Cp had decreasedto below 40% of baseline (FIG. 2). In this strain of mice, there was noanemia observed when the copper decreased to this level. A separategroup of 4 treatment animals was given higher doses of TM between1.0-1.5 mg over 2-4 weeks. The animals who received 1.25-1.5 mg diedafter 1-3 weeks of therapy. Autopsies revealed that one animal died ofaspiration pneumonia (ascribed to gavage accident) whereas 2 animalsdied of renal tubular necrosis and pulmonary hemorrhages, with clearevidence of vascular injury to these tissues on necropsy. These studiessuggest that 1.0 mg/day is the maximum tolerated dose (MTD) for TM inadult mice (average weight 32 grams), when prolonged treatment isplanned.

In order to test whether mammary tumors in the control mice could bedecreased in size with TM, 3 of the control animals were treated afterthe tumors were well established (>1.5 cm largest dimension). In ⅔cases, the tumors were shrunk significantly by 25% and 50%.

Next, the possibility that prolonged TM therapy had somehow damaged thebreast tissue so tumors could not possibly arise in the treatmentanimals due to lack of a target was determined. After 80% of the controlanimals and none of the treatment animals had developed tumors, thecontrol animals were released from treatment. Clinically overt mammarytumors began to develop in this cross-over group of mice (which had beenpreviously disease-free), within 18 days, suggesting that indeed thetumor initiation event in the target tissue had taken place butexpansion of the tumor to a clinically detectable mass was not possiblein the copper deficient state.

Microscopic analyses of the mammary glands of the TM-treated micerevealed a number (1-8) of small “micro-tumors” approximately 3-10cell-layers thick which failed to vascularize. However, upon releasefrom TM therapy, these micro tumors grew very rapidly into palpablemasses that were briskly vascularized. Although not all the cellular andmolecular details have yet been elucidated, it is clear that in thisimportant carcinogenesis model, copper deficiency inhibited theangiogenic switch, or a step closely downstream from it, as none of themicro-tumors of the treated mice were vascularized.

In summary, decreasing copper availability decreases and eventuallyarrests solid tumor growth, including growth of metastases. The decreasein copper availability needed to decrease tumor growth in humans withoutWilson's disease was ascertained as described herein below. The successof TM as an anti-tumor agent is based at least in part on therelationship between the degree of copper deficiency required to obtainefficacy, and the toxicity of that degree of copper deficiency. As tumorangiogenesis is abrogated when mild copper deficiency ensues, TM is aremarkably effective agent for several different oncologic applications.Other settings contemplated for TM therapy include, but are not limitedto, maintenance therapy following chemotherapy or bone marrowtransplantation, in patients who are elderly or otherwise ineligible toreceive chemotherapy, or as a cytostatic agent in high-risk individuals(inflammatory cancer, multiple positive nodes).

C. Nude Mice with Transplanted Tumors

In this study, the ability of tetrathiomolybdate (TM) to abrogate orretard the growth of tumors in the mammary pads of nude mice afterorthotropic injection of human breast cancer cells was studied. Thehighly angiogenic inflammatory breast cancer cell line called SUM149 wasselected for this study. Given its high propensity to form palpabletumors within 2 weeks of injection into the mammary of 80-100% of thenude mice injected, this cell line posed a stringent test of the abilityof TM to impair tumor progression.

Three groups of 5 nude mice each were set up in separate cages. Groups 2and 3 received TM in the drinking water to average an intake of 1.2mg/day/mouse, starting at day −7. Groups 1, 2, and 3 were injected onday 0 with 10⁶ SUM149 breast cancer cells in the second thoracic mammaryfat pad. On day 34, as no palpable tumors were noticeable in any of themice in the treatment groups, TM was skipped on groups 2 and 3, and thenTM was restarted on group 2 at a dose of 0.6 mg/day/mouse, while TM wasresumed at full dose (1.2 mg/mouse/day) in group 3.

The developing tumors were measured approximately twice weekly, and theaverage of the product of the bi-dimensional perpendicular diameters foreach group versus time is plotted in FIG. 3. The tumors in the controlgroup grew relatively rapidly and all animals developed tumors. Incontrast, for the treatment groups, no tumors were palpable until TM wasskipped on day 34. Thereafter, the tumors grew more rapidly in the groupthat received ½ dose of TM, which is known to only decrease the copperlevels by 50%. In contrast, only very small tumors have grown in thegroup treated with TM at fill dose, which is defined as 1.2mg/day/mouse. This dose decreases copper to approximately 10-20% ofbaseline levels. It is this degree of copper deficiency, well toleratedin both mice and humans, which appears to be required to inhibit tumorangiogenesis.

This study supports the idea that TM probably inhibits angiogenesis atleast in 2 ways: one is by inhibition of the “angiogenic switch”, andthe other is by inhibition of bulk tumor angiogenesis. Skipping TM onday 34 enabled the angiogenic switch to activate and begin to bring avessel to the tumor cluster which had been injected in copper deficientnude mice. This activation of the switch enables some tumor growth,although it is clear that the group treated with TM has much slowertumor growth, and appears to be reaching a plateau. This study alsovalidates oral administration of TM in the drinking water of the nudemice.

EXAMPLE 3 Phase I/II Clinical Trial of TM as Anti-Cancer Therapy

A. Introduction

Patients with metastatic solid tumors often have very limited treatmentoptions due to the cumulative toxicity of cytoreductive chemotherapy anddrug resistance. Following the pre-clinical work detailed above, whichshowed efficacy for the anti-copper approach in mouse tumor models, aPhase I clinical trial was conducted in 18 patients with metastaticcancer who were enrolled at 3 dose levels of oral tetrathiomolybdate(TM; 90, 105, and 120 mg/day) administered in 6 divided doses with andin-between meals. Serum ceruloplasmin (Cp) was used as a surrogatemarker for total-body copper. As anemia is the first clinical sign ofcopper deficiency, the goal of the study was to reduce Cp to 20% ofbaseline value, without reducing hematocrit by more than 80% ofbaseline. Cp is a reliable and sensitive measure of copper status, andTM was non-toxic when Cp was reduced to 15-20% of baseline. The levelIII dose of TM of 120 mg/day was effective in reaching the target Cp,without added toxicity. TM-induced mild copper deficiency achievedstable disease in 5 out of 6 patients who were copper deficient at thetarget range for at least 90 days.

1. Toxicity

The pharmacological effects of TM are completely specific to copper. Asit has no detectable effects on other minerals, its toxicity is thendirectly related to copper deficiency. Other than copper deficiency, atthe doses employed, there is no toxicity described in animals. Inhumans, there are two reports of reversible anemia in Wilson's diseasepatients taking TM for maintenance therapy at doses of 30-40 mg 6 timesper day. In treating 45 patients with Wilson's disease for eight weekswith TM as initial therapy for Wilson's disease, five cases ofreversible anemia (11.1%) were observed. The anemia is due to decreasedheme synthesis as a result of copper depletion in the bone marrow. Thepatients whose blood showed the most severe reduction in Cu levelsexhibit this anemia.

As mentioned above, the copper status of an animal or human undergoingTM therapy cannot be followed by serum copper alone, because the complexof TM, copper, and albumin, is cleared more slowly from the blood thanit accumulates, until a high copper steady state is reached. Thiscomplexed copper is however not available for cellular uptake, and isgradually cleared from the body via the urine and the bile, withoutparticipating in any cellular copper-dependent processes, such asangiogenesis. The inventors have developed guidelines for monitoring thecopper balance in humans during TM therapy, according to the recognizedthree stages of copper deficiency (Brewer, 1992; Brewer et al., 1991a),as follows.

First Stage—Chemical Copper Deficiency

During this stage, the serum ceruloplasmin (Cp) activity is decreased upto approximately 5-10% of baseline. Cp, a copper containing protein, issynthesized in the liver and Cp synthesis is decreased during copperdepletion. This stage of copper deficiency, although measurable in thelaboratory, has no clinical signs or symptoms. The Cp must be below afew percent of normal before early clinical copper deficiency ensues.

Second Stage—Mild Clinical Copper Deficiency

After the Cp is held at 0-5% for a period of 5-10 days, the firstclinical signs of copper deficiency may appear. These are mildneutropenia and hypochromic microcytic red cell changes. Both the whiteblood count and hematocrit fall to approximately 75-85% of baseline.Copper is required for heme synthesis, so the morphological changes seenin the peripheral smear are similar to those characteristic of irondeficiency. The anisocytosis and poikilocytosis exacerbate as copperdeficiency becomes more severe. The onset of this mild anemia andneutropenia is gradual and often entirely asymptomatic.

Third Stage—Moderate to Severe Clinical Copper Deficiency

Typically when Hct<70% of baseline, more severe clinical signs andsymptoms resulting from inadequate hematopoesis ensue. These are loss ofappetite, weight loss, diarrhea, impaired melanogenesis with subsequentloss of hair color, rarely cardiac arrhythmias.

The inventors reasoned that mild chemical copper deficiency, (acondition which is extremely well tolerated by humans for seemingly longperiods of time) has efficacy as a strategy to inhibit solid tumorangiogenesis. Modulation and careful monitoring of the degree of copperdeficiency to establish surrogate end-points of efficacy of TM istherefore an important element of this approach.

The inventors have very good evidence-based knowledge about the variousstages of relative copper deficiency, the dividing line between chemicaland clinical copper deficiency, and appropriate protocols on what tomeasure to assess copper status. Since the patients are closelymonitored, the Phase I study has so far proven to be quite safe. Themajor therapeutic issue to be discerned is whether the tumor angiogenicrequirements for copper are significantly higher than essential cellularhousekeeping needs for copper.

2. Pharmacodynamics

TM is administered orally with and without meals, and is well absorbedunder the latter condition. TM forms a tripartite complex with copperand protein, thereby binding copper in food when administered withmeals, preventing copper absorption, or in the bloodstream(TM-Cu-albumin) after absorption, preventing cellular copper uptake. Inpatients with normal copper metabolism, a stoichiometric 1:1relationship between non-ceruloplasmin plasma copper (potentiallyavailable for angiogenesis) and plasma molybdenum is expected, with 6daily doses of 10-20 mg each. The dose level of TM which will result ina steady state of mild copper deficiency varies within the range of40-90 mg daily for most individuals. The complex of TM-Cu-protein isslowly excreted predominantly in bile, with a small amount excreted alsoin the urine. Twenty-four hour urine measurements of Mo and Cu will helpdetermine the rate of elimination of the tripartite complex.

B. Methods

1. Patients

Eighteen adults with metastatic solid tumors, exhibiting measurabledisease, life expectancy of 3 or more months, and at least 60% Karnofskyperformance status (Table 2), were enrolled. Patients with effusions orbone marrow involvement as the only manifestations of disease, and thosewho had severe intercurrent illness requiring intensive management orwere transfusion dependent, were excluded. Patients had to haverecovered from previous toxicities, and had the following requirementsfor laboratory parameters: WBC≧3,000/mm³, ANC≧1,200/mm³, Hct≧27%,Hgb≧8.0 gm/dl, platelet count≧80,000/mm³, bilirubin<2.0 mg/dl, AST/ALT<4times the upper limit of institutional norm, serum creatinine<1.8 mg/dlor calculated creatinine clearance>55 ml/min, calcium≧11.0, albumin≧2.5gm/dl, PT<13 sec., and PTT<35 sec. Other requirements were demonstrableprogression of disease in the previous 3 months, after standardtreatments such as surgery, chemotherapy, radiotherapy, and/orimmunotherapy, or progressive disease after declining conventionaltreatment modalities.

TABLE 2 Karnofsky And Criteria ECOG Performance KARNOFSKY SCORE ECOGACTIVITY % GRADE ACTIVITY Normal, no complaints 100 0 Fully active, ableto carry Normal, only minor 90 on all predisease activitiessigns/symptoms without restrictions Normal activity, but 80 1 Nophysically strenuous requires effort activity, but ambulatory Unable todo active 70 and able to carry out light work, but able to care orsedentary work (e g., for self office work, light house work Able tocare for most 60 2 Ambulatory/capable of all needs, requires self-care,unable to perform occasional help any work activities. Up Requiresfrequent 50 and about medical help and more than 50% of wakingconsiderable assistance hours Disabled, needs 40 3 Capable of onlylimited special care and self-care, confined to bed assistance or chairmore than 50% of Severely disabled, 30 waking hours needshospitalization, death not imminent Very sick, 20 4 Completely disabled,hospitalized, active totally confined to bed or support needed chair.Cannot carry on any self-care. Moribund 10 Dead 0 5 Dead

2. Treatment Schema: Doses and Escalation

Three dose regimens were evaluated. All dose levels consisted of TM 20mg given 3 times daily with meals plus an escalating (levels I, II, andIII) in-between meal dosing, given 3 times daily, for a total of 6 dosesper day. Loading dose levels I, II, and III provided TM at 10 mg, 15 mg,and 20 mg, 3 times daily between meals, respectively, in addition to the3 doses of 20 mg each, given with meals, at all dose levels.

Baseline Cp was taken as the nearest Cp measurement to day 1 oftreatment, including day 1, since the blood was drawn pre-TM, for allpatients. The target Cp reduction was defined as 20% of baseline Cp. Dueto Cp assay variability of approximately 2%, a change of Cp to 22% ofbaseline was considered as achieving the desired reduction of copper. Inaddition, if the absolute Cp was less than 5 mg/dl, then the patient wasconsidered as having reached the target Cp. No patient reached thetarget due to an absolute Cp of less that 5 mg/dl, without also being atleast 78% reduced from baseline. After reaching the target copperdeficient state, TM doses were individually tailored to maintain Cpwithin a target window of 70-90% reduction from baseline.

Six patients were to be enrolled at each dose level. After 4 patientswere enrolled at level I, if one patient experienced dose-limitingtoxicity (DLT) (defined as Hct<80% of baseline), 2 more patients wereenrolled at level I. If no DLT was observed, patients were enrolled atthe next dose level. Treatment was allowed to continue beyond inductionof target copper deficiency, if the patients experienced a partial orcomplete clinical response or achieved clinical stable disease by thefollowing definitions. Complete response is defined as the disappearanceof all clinical and laboratory signs and symptoms of active disease.Partial response is defined as a 50% or greater reduction in the size ofmeasurable lesions defined by the sum of the products of the longestperpendicular diameters of the lesions, with no new lesions or lesionsincreasing in size. Minor response is defined as a 25-49% reduction inthe sum of the products of the longest perpendicular diameters of one ormore measurable lesions, no increase in size of any lesions and no newlesions; stable disease is any change in tumor measurements notrepresented by the criteria for response or progressive disease, whichis defined as an increase of 25% or more in the sum of the products ofthe longest perpendicular diameters of any measurable indicator lesions,compared to the smallest previous measurement or appearance of a newlesion. Because copper deficiency is not a cytotoxic treatment modality,the patients who provide information about the efficacy of TM forlong-term therapy, in this population of patients with advanced cancer,are primarily those who remained within the target Cp window of (20±10)%of baseline for over 90 days, without disease progression

3. Monitoring of Copper Status

A method was required to monitor copper status easily and reliably, sothat TM dose could be adjusted appropriately during this trial. With TMadministration, serum copper is not a useful measure of total-bodycopper, because the TM-copper-albumin complex is not rapidly cleared,and the total serum copper (including the fraction bound to theTM-protein complex) actually increases during TM therapy (Brewer et al.,1991a; 1994b; 1996). The serum ceruloplasmin level obtained weekly wasused as a surrogate measure of total-body copper status. The serum Cplevel is controlled by Cp synthesis by the liver, which, in turn, isdetermined by copper availability to the liver (Linder et al., 1979).Thus, as total-body copper is reduced, the serum Cp level isproportionately reduced. The serum Cp level is in the range of 20-35mg/dl and 30-65 mg/dl for normal controls and cancer patients,respectively. The objective as this trial was to reduce Cp to or below20% of baseline, and to maintain this level, within a window spanned by(20±10) % of the baseline Cp, with typical Cp values in the range of7-12 mg/dl. Since there appears to be no untoward clinical effects fromthis degree of copper reduction, this level of copper deficiency hasbeen termed “chemical copper deficiency”. The first indication of trueclinical copper deficiency is a reduction in blood cell counts,primarily anemia, as copper is required for heme synthesis as well ascellular proliferation (Brewer et al., 1996). Thus, the copperdeficiency objective of this trial was to reduce the Cp to 20% ofbaseline or below, without decreasing the patient's hematocrit or WBC tobelow 80% of the baseline value at entry.

4. Toxicity, Follow-Up, and Disease Evaluation

Complete blood counts, liver and renal function tests, urinalyses, andCp level (by the oxidase method) were performed weekly for 16 weeks,then bi-weekly. Physical examinations and evaluations of toxicity werecarried out every 2 weeks for 8 weeks, then every 4 weeks for theduration of therapy. Toxicity was evaluated using the National CancerInstitute Common Toxicity Criteria (Table 3). As TM is not a cytotoxicdrug at the doses employed, and it has already been given to humanswithout any other toxicities than the ones described in detail above,the majority of the toxicities listed in Table 3, should they arise, arenot expected to be due to TM. Nevertheless, therapy will be discontinuedand the patient removed from the study if grade 3 or higher toxicitiesof any type are observed, whatever the probable etiology. For grade 2toxicities, an attempt will be made to establish their etiology. Ifroutine support care measures do not alleviate the conditions, the drugwill be discontinued and the patients removed from the study.

Extent of disease was evaluated at entry, at the point of achievement ofcopper deficiency, defined as Cp at or below 20% of baseline, and every10-12 weeks thereafter. Computer-assisted tomography or magneticresonance imaging were used as appropriate for conventional measurementof disease at all known sites and for evaluation of any potential newsites of disease. Angiogenesis-sensitive ultrasound with 3-dimensionalDoppler analyses was employed in select cases, as adjunct toconventional imaging, to evaluate the blood flow to the tumors atdifferent time points.

TABLE 3 Toxicity Criteria Toxicity Grade 0 1 2 3 4 Blood/Bone Marrow WBC≧4.0K 3.0-3.9K 2.0-2.9K 1.0-1.9K <1K Platelets WNL 75.0 K-WNL 50-74.9K25.0-49.9K <25K Hemoglobin WNL 10.0 g-WNL 8.0-10.0 g 6.5-7.9 g <6.5 gNeutrophils ≧2.0K 1.5-1.9K 1.0-1.4K 0.5-0.9K <0.5K Lymphocytes ≧2.0K1.5-1.9K 1.0-1.4K 0.5-0.9K <0.5K Hemorrhage, None Mild, No TransfusionsGross, 1-2 U PRBC Gross, 3-4 U PRBC Massive, >4 U PRBC ClinicalInfection None Mild Moderate Severe Life-Threatening GastrointestinalNausea None Able to Eat Intake Decreased No Significant Intake VomitingNone 1 × /24 h 2-5 × /24 h 6-10 × 24/h >10 × /24 h Diarrhea NoneIncrease of 2-3 × /24 h Increase of 4-6 × /24 h Increase of 7-9 × /24 hIncrease of ≧10 × /24 h Stomatitis None Painless Ulcers Painful Ulcers,Can Eat Painful Ulcers, Cannot Eat Requires IV Nutrition HepaticBilirubin WNL <1.5 × WNL 1.5-3.0 × WNL >3 × WNL SGOT/SGPT WNL ≦2.5 × WNL2.6-5.0 × WNL 5.1-20 × WNL >20 × WNL Alk Phos WNL ≦2.5 × WNL 2.6-5.0 ×WNL 5.1-20 × WNL >20 × WNL Liver/Clinical No Change Precoma Hepatic ComaKidney/Bladder Creatinine WNL <1.5 × WNL 1.5-3.0 × WNL 3.1-6.0 ×WNL >6.0 × WNL Proteinuria No Change 1 + <0.3 gm % 2-3 + 0.3-1.0 gm %4 + >1.0 gm % Nephrotic Syndrome Hematuria Negative Microscopic GrossWith Clots Transfusion Alopecia No Loss Mild Total CardiovascularDysrhythmia None Asymptomatic No Persistent No Therapy Requires TherapyHypotension, V-tach/V-fib Therapy Cardiac None Decline of EF by <20%Decline of EF by >20% Mild CHF, Rx Responsive Refractory CHF IschemiaNone Nonspecific ST-T Wave Δ Asymptomatic Ischemic Δ Angina, NoInfarction Acute MI Pericardial None Asymptomatic Effusion Pericarditis,rub, EKG Δ Symptomatic Effusion Tamponade Hypertension None or No ΔTransient, >20 mm Hg Persistent, >20 mm, No Requires TherapyHypertensive Crisis Rx Hypotension None Transient, No Therapy FluidReplacement Hospitalized <48 h Hospitalized >48 h Pulmonary No ChangeAsymptomatic Dyspnea on Exertion Dyspnea, no exertion Dyspnea at RestAbnormal PFT Neurologic Neuro-sensory No Change Mild ParesthesiaModerate Sensory Loss Severe Loss, Symptomatic Neuro-motor No ChangeSubjective Weakness Mild Objective Weakness Impairment of FunctionParalysis Cortical None Mild Somnolence, Moderate Somnolence, Severe,with Confusion or Coma or Seizures Agitation Agitation HallucinationCerebellar None Slight Change Speach Slur Tremor, Ataxia CerebellarNecrosis Coordination Nystagmus Mood No Change Mild Anxiety or ModerateSevere Suicidal Depression Headache None Mild Transient Mod-SevereUnrelenting, Severe Constipation None Mild Moderate Severe Ileus >96 hHearing No Change Asymptomatic Tinnitus Correctable Loss Dear, notCorrectable Audiometry Δ Vision No Change Symptomatic Subtotal LossBlindness Skin No Change Macular/Papular Rash, Rash with PruritusGeneralized Eruption Exfoliative or Ulcerative Asymptomatic Rash AllergyNone Transient Rash, Temp Urticaria, Mild Serum Sickness, Anaphylaxis<38° C. Bronchospasm, T >38° C. Bronchospasm Fever None 37.1-38° C.38.1-40° C. >40°, <24 h <40°, >24 h Local None Pain InflammationPhlebitis Ulceration Plastic Surgery Rx Weight Change <5% 5-9.9%10-19.9% ≧20% Metabolic Hyperglycemia <116 116-160 161-250 251-500 >500,Ketoacidosis Hypoglycemia >64 55-64 40-54 30-39 <30 Amylase WNL <1.5 ×WNL 1.5-2.0 × WNL 2.1-5.0 × WNL >5.1 × WNL Hypercalcemia <10.6 10.6-11.511.6-12.5 12.6-13.5 ≧13.5 Hypocalcemia >8.4 8.4-7.8 7.7-7.0 6.9-6.1 ≦6.0HypoMagnesemia >1.4 1.4-1.2 1.1-0.9 0.8-0.6 ≦5.0 Coagulation FibrinogenWNL .75-1 × WNL .5-74 × WNL .25-.49 × WNL ≦>24 × WNL PT WNL 1-1.25 × WNL1.26-1.5 × WNL 1.51-2.0 × WNL >2.0 × WNL PTT WNL 1-1.25 × WNL 1.26-1.5 ×WNL 1.51-2.0 × WNL >2.0 × WNL

5. TM Preparation and Storage

TM was purchased in bulk lots suitable for human administration (AldrichChemical Company, Milwaukee, Wis.). As TM is slowly degraded whenexposed to air, with oxygen replacing the sulfur in the molecule,rendering it inactive (Brewer et al., 1991a; 1994b; 1996), it was storedin 100-gram lots under argon. At the time a prescription was written,the appropriate dose of TM was placed in gelatin capsules. Previously,it was shown by the inventors that TM dispensed in such capsulesretained at least 90% of its potency for eight weeks (Brewer et al.,1991a). Thus, TM was dispensed to each patient in eight-weekinstallments throughout the trial.

6. Measurement of Blood Flow

Blood flow was measured by ultrasound in select patients with accessiblelesions, at the time they became copper deficient, and at variableintervals of 8-16 weeks thereafter. 3D scanning was performed on a GELogiq 700 ultrasound system, with the 739L, 7.5 MHz linear arrayscanhead. The scanning and vascularity quantification techniques were aspreviously described (Carson et al., 1998; LeCarpentier et al., 1999).

B. Results

1. Patient Characteristics

Eighteen eligible patients, 10 males and 8 females, with 11 differenttypes of metastatic cancer who had progressed through or (in one case)declined other treatment options were enrolled in the trial, in theorder in which they were referred. Six, 5, and 7 patients were enrolledat the 90, 105, and 120 mg/day drug levels, respectively, following theprotocol dose escalation schema. One patient originally assigned to the105 mg level was removed early to pursue cytotoxic chemotherapy, due torapid progression of disease. This same patient was later retreated atthe 120 mg level for a longer duration, and thus is counted only at the120 mg drug level for the analyses. The average age was 59; the averagebaseline Cp was 47.8 mg/dl, which is elevated with respect to normalreflecting the patients' disease status. Table 4 summarizes thepatients' characteristics for each dose level.

TABLE 4 Patient Characteristics Assigned TM Dose (mg) 90 105 120 TotalCharacteristic No. of Patients 6 5 7 18 Sex (M/F) 3/3 1/4 6/1 10/8 Age[Mean (SD)] 64 (12) 60 (12) 53 (17) 59 (14) Primary Tumor: Breast 2 2 04 Colon 0 1 0 1 Lung 1 0 0 1 Melanoma 0 0 1 1 Pancreas 0 1 0 1 Prostate2 0 0 2 Angiosarcoma 0 0 2 2 Chondrosarcoma 0 1 0 1 Nasopharyngeal 0 0 11 Hemangioendothelioma 0 0 1 1 Renal 1 0 2 3 Baseline: Baseline Cp mean52.6 49.3 42.7 47.8 Baseline Cp range 36.6-74.1 38.1-65.0 31.9-52.731.9-74.1 Baseline Hct mean 31.9 37.2 41.9 37.3 Baseline Hct range26.6-35.6 33.8-42.5 35.4-45.7 26.6-45.7

2. Toxicity

There were no cardiac, pulmonary, GI, renal, hepatic, hematologic,infectious, skin, mucosal, or neurologic toxicities observed for Cplevels at or above 20% of baseline. Mild (greater than 80% of baselineHct) reversible anemia was observed in 4 patients for Cp levels between10-20% of baseline. Two of these patients had been treated withcytotoxic chemotherapy and two patients had evidence of extensive bonemarrow involvement with their disease at the time of entry into thetrial. Although in the latter two of these cases, the anemia was mostlikely due to causes other than treatment, TM was discontinuedtemporarily until Hct was restored to acceptable levels with transfusionof 2 units of packed RBCs. In one patient, it is very likely that thecopper deficiency caused by TM produced the anemia. Stopping the drugallowed the hematocrit to recover within 5-7 days without the need fortransfusion; at the patient's request, TM was restarted at a lower dose,without further complications of anemia. Several patients experiencedtransient, occasional sulphur-smelling burping, within 30 minutes of TMingestion. No additional toxicities of any type were observed withlong-term maintenance of mild clinical copper deficiency over 8-15months. Of note, no evidence of GI or other mucosal bleeding or impairedhealing of minor trauma were observed with long-term therapy. Onepre-menopausal patient with extensive metastatic renal cancerexperienced normal menstrual periods during TM therapy, including 2.5months of observation while copper deficient with Cp<20% of baseline.

3. Ceruloplasmin as a Surrogate Measure of Copper Status

FIG. 4A, FIG. 4B and FIG. 4C shows the response of Cp as a function oftime on TM therapy, expressed as the ratio of Cp at time t to thebaseline Cp level for each patient enrolled at the 90 mg/day (FIG. 4A),105 mg/day (FIG. 4B), and 120 mg/day (FIG. 4C) dose levels. Increasingthe in-between meals dose from 10 mg 3× daily to 15 mg or 20 mg 3× dailyhad no significant effect on the rate of decrease of the Cp level,reaching a level of 50% baseline at a mean of 30 days (median=28 days).The response of Cp to TM therapy as a function of time exhibited onlyminor fluctuations; when TM was discontinued, a rapid rise in Cp, within48 hours, was observed.

Four patients were removed from study due to progression of diseaseprior to achieving target Cp of 20% of baseline, while the remaining 14patients achieved the target Cp level. Since all 14 patients whoachieved the target Cp level wished to remain on study, they wereallowed to do so, according to the protocol, as long as they did notexhibit disease progression or toxicity. The TM doses were adjusted inthese patients to maintain the Cp between 10-20% of baseline. Thesepatients provide the preliminary evidence of efficacy and of long-termtolerance of this approach.

4. Dose Adjustments to Maintain Target Cp

In order to maintain a Cp target of 20% of baseline and to preventabsolute Cp values less than 5 mg/dl, TM doses were adjusted. Due to aroutine 7-day turn-around for the Cp test, these dose changes were madeapproximately 7-10 days after the blood for the Cp measurement wastaken. After achieving the target Cp, the in-between meals dose wastypically decreased by 20 mg. Further decreases of 15-30 mg werenecessary during long-term therapy. A patient with metastaticchondrosarcoma secondary to radiation treatment for breast cancer onlong-term therapy has stable disease after 12 months of copperdeficiency, with stable quality of life. One biopsy-proven metastaticnodule on the third digit is easily measurable and has been stable.Interestingly, this patient has required only a minor adjustment to theTM dose from the initial loading dose level to maintain the target Cpthroughout this relatively long period.

FIG. 5A and FIG. 5B illustrate the Cp response to dose adjustmentsrequired for two more representative patients over approximately 100days of therapy. The patient in FIG. 5A has so far required onlydecreases in dose 60 days apart. Most patients have required bothincrease and decrease in dose, during long-term therapy. For example, asshown in FIG. 5B, the TM dose was increased after day 100 to respond toan increase in Cp outside the target range. Overall, there wasconsiderable individual variability in the dose adjustments required.Other sites of suspected disease in the chest also remain stable. Inconclusion, the Cp response to TM therapy evaluated weekly is notbrittle or subject to wide fluctuations.

5. Measurement of Response of Metastatic Cancer to TM

a. Clinical Evaluation

Although the patients received different initial loading doses of TM,the Cp maintenance window of (20±10)% of baseline was used in allgroups, regardless of the loading dose. Patients who maintained thisdegree of copper deficiency through tailored adjustments of the TM dosefor over 90 days, are likely to reflect the anti-angiogenic activity ofTM against their tumors. The period of 90 days is selected for 2 mainreasons. First, TM is not cytotoxic to either cancer or endothelialcells and mainly impairs endothelial cell function and pro-angiogenicfactor production. This mechanism of action is expected to have a veryslow effect on the size of tumor masses. Second, as tumors sequestercopper, the microenvironment of the tumor is expected to take a longertime to be rendered copper deficient. Table 5 summarizes the clinicalcourse of the 18 patients.

TABLE 5 Summary of Type and Length of Response to TM Therapy No. ofDuration of Cu Deficiency, days Type of Response Patients (Average) DidNot Achieve Target Cp 4 Achieved Target Cp 14  Target Cp <90 days  8/14Disease progression 7 Stable disease with partial 1  49+ regression oflung lesions Target Cp >90 days  6/14 Stable disease with partial 1/6120+ regression of lung lesions Stable disease 4/6 159*, 329+, 351+,413+ (313+) Disease progression at one 1/6 120+ site, stable elsewhere*Patient discontinued therapy; +On therapy

Fourteen patients achieved the target copper deficiency prior to diseaseprogression or other disease complications. Of these, 8 patients eitherprogressed within 30 days of achieving copper deficiency or have hadstable disease for fewer than 90 days; it is unlikely that most of thesetumors experienced an anti-angiogenic environment long enough toevaluate clinical response to this type of therapy. In all patients whowere removed from protocol due to disease progression or choice, and inone patient due to the need for abdominal surgery to relieve a smallbowel obstruction, much more rapid rates of progression of disease werenoted clinically, after discontinuation of TM therapy.

The remaining 6 patients experienced stable disease (5/6) or progressionof disease at one site, with stable disease elsewhere (1/6). Twopatients who have stable disease by standard criteria also experiencedcomplete disappearance of some lung lesions and decrease in size ofother lung lesions during observation periods at target Cp of 120 and 49days. The 5 patients on long-term (over 90 days) maintenance therapywith stable disease have been copper deficient between 120 and 413 daysat the time of this analysis.

b. Radiologic Evaluation

Serial evaluations of tumor masses by conventional imaging with CAT scanor MRI revealed that the radiographic appearance of the certain masseschanged significantly over time. In particular, areas of presumedcentral necrosis (corresponding to lower attenuation of the X-raysignal) were observed in a variety of tumor types, most notably renalcell cancer, angiosarcoma, and breast cancer. Seeking to evaluate theblood flow to the tumors as a function of time during copper deficiencyon long-term TM therapy, lesions accessible to ultrasound were imagedwith color flow 3-dimensional ultrasound at the onset of copperdeficiency, and at intervals of 2-4 months thereafter.

Conventional CAT scan images and blood-flow sensitive 3D-ultrasound werecompared in a rib metastasis from a renal cell carcinoma upon reachingtarget copper deficiency and 8 weeks later. A CAT scan showed thislesion to be of a stable size over time, although a more distinct regionof (probable) central necrosis is observed 8 weeks after reaching targetcopper deficiency. In comparison, a decrease in blood flow to this massby 4.4-fold in this period of approximately 8 weeks was detected by the3D-ultrasound. In addition to the mass studied by these two techniques,this patient had extensive disease in the chest, pelvis, and femurs.

c. TM in Combination with Other Treatment Modalities

During the long-term maintenance of copper deficiency, additionaltreatment modalities were added to TM, as deemed appropriate for theoptimal management of the patients. A patient with previously untreatedmetastatic breast cancer is doing well with a good to excellent qualityof life after 12 months of treatment. This patient had metastases in theparatracheal, posterior cervical, and retroperitoneal lymph node chains,but had declined all cytotoxic therapy. The patient had stable diseasefor over 6 months on TM treatment, when, due to slight increase (lessthan 25% of baseline) in the bidimensional size of the paratracheal andretroperitoneal nodes, was begun on concurrent trastuzumab therapy,after this drug became commercially available. This patient showed arapid response to trastuzumab at all sites of disease: after 1 cycle,there was a clinical complete response in the neck, and after 3 cyclesof trastuzumab, there was radiologic confirmation of complete responseat all previous sites of disease. The patient remains on TM, but thetrastuzumab was discontinued after 6 doses. The patient continues tomaintain status as a complete responder on TM alone for 3 months afterdiscontinuation of trastuzumab therapy. Because the complete responsewas achieved after addition of trastuzumab therapy, this patient isclassified as having only stable disease on TM in Table 5.

Two patients with extensive angiosarcoma of the face and scalp achievedstable disease on TM. In one patient with severe chronic bleeding froman ocular lesion which threatened the orbit, interferon-alpha 2 (IFN-α)was added to TM to attempt to enhance tumor response. Given thesuggestion that, based on studies of progressing hemangiomas, the use oflow-dose interferon may be efficacious for the treatment of hemangioma(Takahashi et al., 1994), IFN-α was administered to both of thesepatients at a dose of 500,000 units subcutaneously twice a day.Radiotherapy was also given to these 2 patients while on TM, to attemptto control actively bleeding (but not progressing) lesions. Bothpatients had disease stabilization for over 60 days, with one of themremaining with stable disease for over 5 months, prior todiscontinuation of therapy due to patient choice. No exacerbation oftoxicity was observed by addition of any of these treatment modalitiesto TM.

This is the first human trial of induction and maintenance of copperdeficiency with tetrathiomolybdate as an anti-angiogenic therapy forcancer. In a group of patients with advanced cancer, it was demonstratedthat TM is remarkably non-toxic when Cp is lowered to 10-20% of baselinelevels for up to 17 months of treatment. The only drug-related toxicityobserved was mild anemia in one patient, which was easily reversiblewith adjustment in the TM dose to bring the Cp level to the desiredtarget. In spite of the diverse roles that copper plays in diverseessential biological processes including heme synthesis, superoxidedismutase and cytochrome function, no lasting significant adverseeffects were observed upon reduction of Cp to approximately 20% ofbaseline. This level of copper reduction constitutes the lower limit ofchemical copper deficiency and the beginning of mild clinical copperdeficiency, the first manifestation of which is mild anemia.

The use of serum Cp level obtained by the oxidase method, an inexpensiveand widely available test, was validated as a sensitive and reliablesurrogate marker of total-body copper status during TM therapy. Usingthe six times per day dose regimen, and initial TM doses ranging from 90to 120 mg per day, the serum Cp was reliably lowered to 50% of baselinein 17 out of 18 patients treated and to 20% of baseline in 14 out of 18patients. Reduction to 50% of baseline was achieved on the average in 30days, with further reduction to Cp levels of 5-10 mg/dl taking 20-30days. Although this rate of decrease in Cp is reasonable for the initialtreatment of early malignant lesions or in the adjuvant setting, inwidely metastatic advanced cancer, this rate of decrease will beaccelerated to prevent some disease progression during induction ofcopper deficiency in a significant number of patients. Since loadingdose variations of 90 to 120 mg per day do not appear to affect the rateof Cp reduction, and given the typical daily intake of copper with food,higher doses in-between meals will be required to accelerate the rate ofinduction of copper deficiency.

As the Cp response to TM-induced copper deficiency is monotonic andexhibits little inter-subject variability, there is essentially no riskof sudden changes or unpredictable fluctuations that might make dosemanagement difficult. Following Cp levels once every one to two weeks isadequate to monitor copper status. As a corollary, overtreatment iseasily detectable and correctable.

As a result of this study it is apparent that, with the present TM doseregimens, there is considerable lag between initiation of TM therapy andreduction of copper levels in tumors to a likely anti-angiogenic level.Further retarding the ability to reach anti-angiogenic levels of copperdeficiency is the likelihood that most tumors sequester copper (Arnoldand Sasse, 1961; Apelgot et al., 1986; Gullino et al., 1990; Fuchs andSacerdote de Lustig, 1989). Thus, additional time may be required todeplete the tumor micro-environment to an effectively low level ofcopper, defined as a level low enough to inhibit angiogenesis. Patientswith very rapidly progressive large tumors may therefore requireadditional treatment modalities, as described herein, in addition toanti-angiogenesis therapy.

Furthermore, initially effective anti-angiogenesis may cause brisk tumornecrosis, resulting in the release of additional copper from the dyingcells. In the case of one patient, a transient rise in Cp was observedat approximately the same time as the ultrasound suggested that thelarge tumor mass might be undergoing central necrosis due to asignificant decrease in blood flow. Thus, a period of 60-90 days of Cpat the target level of 20% of baseline is a reasonable starting pointfor evaluation of response to anti-copper therapy. In the two patientswho exhibited partial regression of lung lesions, tumor control may havebegun earlier. It is also interesting to note that, in both of thesepatients, the lung parenchymal metastases were the sites of tumorregression. It is possible that mild clinical copper deficiency impairssuperoxide dismutase function (Culotta et al., 1997) so that underconditions of high oxidant stress, such as those present in the lung,the metastatic foci are more susceptible to oxidative damage.

In spite of individual differences, the use of 3D-ultrasound todetermine the total blood flow to a given mass demonstrates thatmaintenance of mild copper reduction to 20% of baseline induced for atleast 8 weeks appears sufficient to alter tumor blood flow. Due to therelative insensitivity of computer-assisted tomography to the blood flowor metabolic status of the lesions, parallel imaging modalities, asdemonstrated here for 3-D ultrasound, are preferred to assess functionalresponse in addition to tumor size.

These studies show that that the size of solid tumors of a variety oftypes may be stabilized or decreased by TM, given sufficient time in astate of mild clinical copper deficiency represented by a decrease in Cpto or below 20% of baseline, as defined by this study. Among thepatients who were maintained at the target Cp level for more than 90days, a significant proportion of cases (5/6) were stabilized, with nodetriment to their quality of life. In this population of patients withadvanced disease, 39% of those treated were able to be maintained at thetarget Cp for this duration.

The pattern and speed of progression observed in these patients havealso provided useful information. One patient achieved stable disease atall sites but one, and has chosen to remain on TM therapy due to diseasestabilization at the more life-threatening sites of disease (bowel andparatracheal lymph nodes). Interestingly, the site of progression inthis patient with melanoma is a large adrenal metastasis, which is atpresent being irradiated. This and other observations in this trialsuggest that, whereas copper deficiency may be generally inhibitory ofangiogenesis, heterogeneity of tumor type and the specific location ofmetastases may modulate the response to this therapeutic modality. Sinceit appears that lesions progress at a much faster rate upon copperrepletion than while on TM therapy, adjunct modalities, eithersystemically or local-regionally, may be used to address the specificsites of progression, while allowing the patients to remain in a copperdeficient state.

These studies also show that combination therapies of TM withradiotherapy, trastuzumab, and interferon-alpha, occur without apparentexacerbation of toxicity of the added modality. Taken as a whole, thesafety and preliminary efficacy data derived from this trial supportsthe use of TM alone, or in combination, for the treatment of earlymetastatic disease, minimal disease, and in adjuvant high-risk clinicalsettings, including chemoprevention.

EXAMPLE 4 Phase II/III Clinical Trial of TM as Anti-Cancer Therapy

A. Introduction

Four considerations enter into designing the drug dose and schedule forthis trial. The first is that the dose regimens previously used,although effective in reducing copper, take too long to get to the Cpendpoint in order to properly evaluate efficacy. As it is known thattumors sequester copper (Apelgot et al., 1986; Arnold and Sasse, 1961),anti-angiogenic effects will not likely be detected until systemiccopper deficiency has been maintained perhaps for at least a few weeksto months. Thus, it is important to get to the endpoint of systemiccopper deficiency (0-20% Cp level) as quickly as possible to maximizethe potential for efficacy. Thus, the present trial utilizes a “loading”dose, to be used for two weeks, to achieve Cp criteria, followed by alower maintenance dose, to remain at the target Cp of 0-20% baseline.This design utilizes the knowledge gained in the Phase I trial and willdetermine the efficacy of TM to provide stable disease or tumordecrease.

Second, the trial will evaluate whether an efficacious response dependson how rigorously Cp levels are controlled. Thus, in the first group ofpatients, Cp levels are maintained between 10 and 20%, and in the secondgroup Cp levels are maintained between 0 and 10%.

Third, this trial will determine whether maintenance of a low copperstatus is more easily maintained with zinc therapy. The inventors havedeveloped zinc as an FDA approved therapy for Wilson's disease. It actsby inducing intestinal metallothionein and blocking the absorption ofcopper. Thus in 6 patients, the low copper status will be maintained byusing 25 mg of zinc tid, with dose adjustments as necessary to maintainCp criteria.

B. Phase II Study

1. Loading Dose

The inventors have found that a dosing schedule of 20 mg tid with meals,and a single dose of 60 mg between meals reduces the Cp level to <20%more rapidly than 20 mg tid with meals and 20 mg tid between meals.Therefore, this dosing schedule will be studied in a Phase II study.Three other loading doses of TM will also be studied: Level 1: 20 mg tidbetween meals and 20 mg tid with meals until Cp<20% (10 patients); Level2: 25 mg tid between meals and 25 mg tid with meals until Cp<20% (10patients); and Level 3: 30 mg tid between meals and 30 mg tid with mealsuntil Cp<20% (10 patients).

The objective of the loading dose study is to arrive at the desired Cplevel (<20% of baseline) within 2-3 weeks. The trial will begin withLevel 1. If Level 1 achieves the desired Cp level, all 30 patients willbe loaded at dose Level 1. If Level 1 does not achieve the desired Cplevel, the trial will move to loading dose Level 2, and on to dose Level3 if necessary. Each of these doses are safe for weeks to a few months.

2. Maintenance Dose of TM

Two levels of maintenance dose will be studied: Level 1: TM dosesadjusted as necessary to maintain Cp at 10-20% of baseline (12patients); and Level 2: TM doses adjusted as necessary to maintain Cp at0-10% of baseline (12 patients).

The objective of comparing two maintenance dose levels is to get ageneral picture of whether more stringent copper control tends toenhance efficacy. In general, the tumor types will be randomized betweenthe two levels.

3. Maintenance Dose of Zinc

Two patients from each loading dose group, for a total of six patients,will be treated with zinc therapy for maintenance control. The trialwill begin with 25 mg of zinc 3 times per day (away from food) andadjust the dose to maintain Cp below 20% of baseline.

The objective of the zinc study is to see if it is easier to controlcopper status during maintenance with zinc than with TM, and to get ageneral picture of whether efficacy with zinc is generally comparable toefficacy with TM.

4. Patient Selection Criteria

The patient selection criteria will include: a) metastaticadenocarcinoma, squamous carcinoma, or sarcoma of any organ of origin;b) measurable disease by chest X-ray, CAT Scan, or plain bone films; c)progressive disease documented at least once within 3 months of entry;d) ability to provide informed consent; e) performance status ECOG 0-1;and f) life expectancy ≧6 months.

Exclusion criteria include hematocrit less than 29, LFT's more than fourtimes normal, or severe concurrent medical disease requiring intensivemanagement.

5. Parameters

The parameters that will be followed in the patients are: 1) CBCplatelets, weekly; 2) electrolytes, BUN, creatinine, LFT's weekly; 3)blood for serum copper, molybdenum, ceruloplasmin weekly; 4) urinalysisweekly; 5) clinical status every 2 weeks; 6) tumor measurement every 4weeks; and 7) research angiogenesis—sensitive ultrasound every 8 weeks.

6. Toxicity Endpoints

As before, the drug will be stopped when either hematocrit or WBC dropsbelow 80% of baseline. The drug will also be stopped if there isevidence of possible systemic toxicity, such as abnormal liver or renalfunction tests, or any other grade 3 or higher toxicity by NCI criteria.

7. Length of Study

If there is not evidence of disease stabilization or reduction in apatient by 6 months, the study in that patient will be terminated.Otherwise, therapy will continue as long as the disease is controlledwithin 25% of the initial size at all sites, there is not significanttoxicity, and the patient desires to continue.

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods, and in the steps or in the sequence ofsteps of the methods described herein, without departing from theconcept, spirit and scope of the invention. More specifically, it willbe apparent that certain agents that are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

-   U.S. Pat. No. 5,399,586, issued Mar. 21, 1995.-   U.S. Pat. No. 5,464,833, issued Nov. 7, 1995.-   U.S. Pat. No. 5,504,074, issued Apr. 2, 1996.-   U.S. Pat. No. 5,539,094, issued Jul. 23, 1996.-   U.S. Pat. No. 5,565,491, issued Oct. 15, 1996.-   U.S. Pat. No. 5,571,523, issued Nov. 5, 1996.-   U.S. Pat. No. 5,576,330, issued Nov. 19, 1996.-   U.S. Pat. No. 5,583,034, issued Dec. 10, 1996.-   U.S. Pat. No. 5,587,459, issued Dec. 24, 1996.-   U.S. Pat. No. 5,591,717, issued Jan. 7, 1997.-   U.S. Pat. No. 5,593,664, issued Jan. 14, 1997.-   U.S. Pat. No. 5,599,813, issued Feb. 4, 1997.-   U.S. Pat. No. 5,605,826, issued Feb. 25, 1997.-   U.S. Pat. No. 5,618,925, issued Apr. 8, 1997.-   U.S. Pat. No. 5,622,829, issued Apr. 22, 1997.-   U.S. Pat. No. 5,639,725, issued Jun. 17, 1997.-   U.S. Pat. No. 5,650,491, issued Jul. 22, 1997.-   U.S. Pat. No. 5,654,155, issued Aug. 5, 1997.-   U.S. Pat. No. 5,672,603, issued Sep. 30, 1997.-   U.S. Pat. No. 5,677,178, issued Oct. 14, 1997.-   U.S. Pat. No. 5,693,473, issued Dec. 2, 1997.-   U.S. Pat. No. 5,693,627, issued Dec. 2, 1997.-   U.S. Pat. No. 5,709,999, issued Jan. 20, 1998.-   U.S. Pat. No. 5,710,001, issued Jan. 20, 1998.-   U.S. Pat. No. 5,716,981, issued Feb. 10, 1998.-   U.S. Pat. No. 5,733,876, issued Mar. 31, 1998.-   U.S. Pat. No. 5,747,282, issued May 5, 1998.-   U.S. Pat. No. 5,747,469, issued May 5, 1998.-   U.S. Pat. No. 5,750,400, issued May 12, 1998.-   U.S. Pat. No. 5,750,653, issued May 12, 1998.-   U.S. Pat. No. 5,753,441, issued May 19, 1998.-   U.S. Pat. No. 5,756,294, issued May 26, 1998.-   U.S. Pat. No. 5,756,455, issued May 26, 1998.-   U.S. Pat. No. 5,763,223, issued Jun. 9, 1998.-   U.S. Pat. No. 5,776,704, issued Jul. 7, 1998.-   U.S. Pat. No. 5,776,743, issued Jul. 7, 1998.-   Abrams and Oldham, Monoclonal Antibody Therapy of Human Cancer, Foon    and Morgan (eds.), Martinus Nijhoff Publishing, Boston, pp. 103-120,    1985.-   Allen and Solomons, “Normal Intestinal Mechanisms in the Absorption    of Copper,” In: Absorption and Malabsorption of Mineral Nutrients,    Solomons and Rosenberg (Eds), Alan R. Liss, Inc., New York, 12:206,    1984.-   Apelgot, Coppey, Fromentin, Guille, Poupon, Roussel, “Altered    Distribution of Copper (⁶⁴Cu) in Tumor-Bearing Mice and Rats,”    Anticancer Research 6:159-164, 1986.-   Arnold and Sasse, “Quantitative and Histochemical Analysis of Cu,    Zn, and Fe in Spontaneous and Induced Primary Tumors of Rats,”    Cancer Res. 21:761-766, 1961.-   Badet, Soncin, Guitton, Lamare, Cartwright and Barritault, “Specific    binding of angiogenin to calf pulmonary artery endothelial cells,”    Proc. Natl. Acad. Sci. USA 86:8427-8431, 1989.-   Baxter, et al., “Transport of fluid and macromolecules in    tumors. III. Role of binding and metabolism,” Microvasc. Res.    41:5-23, 1991.-   Benjamin, Golijanin, Itin, Pode and Keshet, “Selective ablation of    immature blood vessels in established human tumors follows vascular    endothelial growth factor withdrawal,” J. Clin. Invest. 103:159-165,    1999.-   Bickel, Neale, Hall, “A Clinical and Biochemical Study of    Hepatolenticular Degeneration (Wilson's Disease),” Quart. J. Med.    50:527, 1957.-   Borgstom, Bourdon, Hillan, Sriramarao and Ferrara, “Neutralizing    anti-vascular endothelial growth factor antibody completely inhibits    angiogenesis and growth of human prostate carcinoma micro tumors in    vivo,” Prostate 35:1-10, 1998.-   Borgstom, Hillan, Sriramarao and Ferrara, “Complete inhibition of    angiogenesis and growth of microtumors by anti-vascular endothelial    growth factor neutralizing antibody: Novel concepts of angiostatic    therapy for intravital videomicroscopy,” Cancer Res. 56:4032-4039,    1996.-   Brem, Tsanaclis, Zagzag, “Anticopper Treatment Inhibits Pseudopodial    Protrusion and the Invasive Spread of 9L Gliosarcoma Cells in the    Rat Brain,” Neurosurgery 26:391-396, 1990a.-   Brem, Zagzag, Tsanaclis, Gately, Elkouby, Brien, “Inhibition of    angiogenesis and tumor growth in the brain. Suppression of    endothelial cell turnover by penicillamine and the depletion of    copper, an angiogenic colactor,” Am. J. Pathol., 137(5):1121-1142,    1990b.-   Bremner and Young, “Effects of Dietary Molybdenum and Sulphur on the    Distribution of Copper in Plasma and Kidneys of Sheep,” Br. J. Nutr.    39:325, 1978.-   Bremner, Mills, Young, “Copper metabolism in rats given di- or    trithiomolybdates,” J. Inorg. Biochem., 16: 109, 1982.-   Brewer and Yuzbasiyan-Gurkan, “Wilson Disease,” Medicine,    71(3):139-164, 1992a.-   Brewer and Yuzbasiyan-Gurkan, “Wilson's Disease,” In: Textbook of    Clinical Neuropharmocology and Therapeutics, 2nd Edition, Klawans,    Goetz, Tanner (Eds.), Raven Press, New York, pp. 191-205, 1992b.-   Brewer and Yuzbasiyan-Gurkan, “Wilson's disease: an update, with    emphasis on new approaches to treatment,” Dig. Dis., 7(4):178-193,    1989.-   Brewer, “Interactions of zinc and molybdenum with copper in therapy    of Wilson's disease,” Nutr., 11(1 Suppl):114-116, 1995b.-   Brewer, “Practical recommendations and new therapies for Wilson's    disease,” Drugs, 50(2):240-249, 1995a.-   Brewer, “Thiomolybdates in the treatment of Wilson's disease,”    Letter to the Editor. Arch. Neurol., 49: 132-133, 1992.-   Brewer, “Zinc in the Treatment of Wilson's Disease,” Nutrition and    the MD. 19(12): 1993.-   Brewer, Dick, Johnson, Wang, Yuzbasiyan-Gurkan, Kluin, Fink, Aisen,    “Treatment of Wilson's disease with ammonium tetrathiomolybdate. I    Initial therapy in 17 neurologically affected patients,” Arch.    Neurol., 51(6):545-554, 1994b.-   Brewer, Dick, Schall, Yuzbasiyan-Gurkan, Mullaney, Pace, Lindgren,    Thomas, Padgett, “Use of Zinc Acetate to Treat Copper Toxicosis in    Dogs,” JAVMA 201:564-568, 1992a.-   Brewer, Dick, Yuzbasiyan-Gurkan, Johnson, Wang, “Treatment of    Wilson's Disease with Zinc XIII: Therapy with Zinc in Presymptomatic    Patients from the Time of Diagnosis,” J. Lab. Clin. Med.    123:849-858, 1993d.-   Brewer, Dick, Yuzbasiyan-Gurkan, Tankanow, Young, Kluin, “Initial    Therapy of Wilson's Disease Patients with Tetrathiomolybdate,” Arch.    Neurol. 48(1):42-47, 1991a.-   Brewer, Hill, Dick, Nostrant, Sams, Wells, Prasad, “Treatment of    Wilson's Disease with Zinc III. Prevention of Reaccumulation of    Hepatic Copper,” J. Lab. Clin. Med. 109:526-531, 1987b.-   Brewer, Hill, Prasad, Cossak, Rabbini, “Oral zinc therapy for    Wilson's disease,” Annals Int. Med., 99:314-320, 1983.-   Brewer, Hill, Prasad, Dick, “Treatment of Wilson's Disease with    Zinc: IV. Efficacy Monitoring using Urine and Plasma Copper,” Proc.    Soc. Exper. Biol. Med. 7:446-455, 1987c.-   Brewer, Johnson, Dick, Kluin, Fink, Brunberg, “Treatment of Wilson's    disease with ammonium tetrathiomolybdate II. Initial therapy in 33    neurologically affected patients and follow-up on zinc therapy,”    Arch. Neurol. 53:1017-1025, 1996.-   Brewer, Schall, Dick, Yuzbasiyan-Gurkan, Thomas, Padgett, “The Use    of ⁶⁴Copper Measurements to Diagnose Canine Copper Toxicosis,” J.    Vet. Int. Med. 6:41-43, 1992b.-   Brewer, Terry, Aisen, Hill, “Worsening of Neurological Syndrome upon    Initial Treatment of Wilson's Disease Patients with Penicillamine,”    Arch. Neurol. 44:490-494, 1987a.-   Brewer, Turkay, Yuzbasiyan-Gurkan, “Development of neurologic    symptoms in a patient with asymptomatic Wilson's disease treated    with penicillamine,” Arch. Neurol. 51:304-305, 1994a.-   Brewer, Yuzbasiyan-Gurkan, Dick, “Zinc Therapy of Wilson's    Disease VIII. Dose Response Studies,” J. Trace Elem. Exp. Med.    3:227-234, 1990.-   Brewer, Yuzbasiyan-Gurkan, Johnson, “Treatment of Wilson's Disease    with Zinc: IX. Response of Serum Lipids,” J. Lab. Clin. Med.    118:466-470, 1991b.-   Brewer, Yuzbasiyan-Gurkan, Johnson, Dick, Wang, “Treatment of    Wilson's Disease with Zinc XI. Interaction with other Anticopper    Agents,” J. Amer. Coll. Nut. 12(1), 26-30, 1993a.-   Brewer, Yuzbasiyan-Gurkan, Johnson, Dick, Wang, “Treatment of    Wilson's Disease with Zinc XII. Dose Regimen Requirements,” Amer. J.    Med. Sci. 305: (4),199-202; 1993b.-   Brewer, Yuzbasiyan-Gurkan, Lee, “Regulation of Copper Balance and    Its Impairment in Man and Dog,” In: Essential and Toxic Trace    Elements in Human Health and Disease: An Update, Prasad (Ed.),    Allan R. Liss, New York, PCBR 380:129-145, 1993c.-   Brewer, Yuzbasiyan-Gurkan, Lee, Appelman, “Treatment of Wilson's    Disease with Zinc VI. Initial Treatment Studies,” J. Lab. Clin. Med.    114: 33-638, 1989.-   Brewer, Yuzbasiyan-Gurkan, Young, “Treatment of Wilson's Disease,”    Sem. Neurol. 7:209-220, 1987d.-   Byers and Baldwin, “Therapeutic strategies with monoclonal    antibodies and immunoconjugates,” Immunology 65:329-335, 1988.-   Carson, Fowlkes, Roubidoux, Moskalik, Govil, Normolle, LeCarpentier,    Nattakom and Helvie, “3-D Color Doppler image quantification of    breast masses,” Ultrasound Med. Biol. 24:945-952, 1998.-   Coucouvanis et al., “An inorganic functional group approach to the    systematic synthesis and reactivity studies of binuclear Mo/S and    Mo/S/O complexes,” Polyhedron 8:1705-1716, 1989.-   Coucouvanis et al., “Dinuclear Fe—Mo—S complexes containing the    FeS2Mo core. Syntheses, ground-state electronic structures, and    crystal and molecular structures of the    [(C₆H₅)₄P]₂[(C₆H₅S)₂FeS₂MoS₂], [(C₂H₅)₄N]₂[(C₆H₅S)₂FeS₂WS₂], and    [(C₆H₅)₄P]₂[(S)₅FeS₂MS₂] (M=Mo, W) complexes,” Inorg. Chem.    22:293-308, 1983.-   Coucouvanis et al., “Heterodinuclear Di-μ-sulfido bridged dimers    containing iron and molybdenum or tungsten. Structures of    (PhP)₂(FeMS₉) complexes (M=Mo, W),” J. Am. Chem. Soc. 102:1730-1732,    1980a.-   Coucouvanis et al., “Successful isolation of a reduced    tetrathiometallate complex. Synthesis and structural    characterization of the [(MoS₄)₂Fe]³⁻ trianion,” J. Am. Chem. Soc.    102:6644-6646, 1980b.-   Coucouvanis et al., “Synthesis and structural characterization of    [(No)₂FeS₂MoS₂]²⁻ a dinitrosyl complex containing the FeS₂MoS₂    core,” Inorg. Chim. Acta 53:L135-L137, 1981.-   Coucouvanis et al., “Synthesis of thiomolybdenyl complexes with    [Mo₂(S)₂(O)₂]²⁺ cores and substitutionally labile ligands. Crystal    and molecular structure of the [Mo₂O₂S₄(DMF)₃] complex,” Inorg.    Chem. 27:3272-3273, 1988.-   Coucouvanis et al., “Trinuclear Fe-M-S complexes containing a linear    Fe-M-Fe array and a bridging S2MS2 unit. Electronic structures and    crystal and molecular structures of the [(C₆H₅)₄P]₂[Cl₂FeS₂MS₂FeCl₂]    (M=Mo, W) complexes,” Inorg. Chem. 23:741-749, 1984.-   Coucouvanis, “Fe-M-S complexes derived from MS₄ ²⁻ anions (M=Mo, W)    and their possible relevance as analogues for structural features in    the Mo site of nitrogenase,” Acc. Chem. Res. 14:210-209, 1981.-   Coucouvanis, “Syntheses, structures, and reactions of binary and    tertiary thiomolybdate complexes containing the (O)Mo(S_(x)) and    (S)Mo(S_(x)) functional groups (x=1, 2, 4),” Adv. Inrog. Chem.    45:1-73, 1998.-   Cox, Davis, Shirley, Jack, “Influence of excess dietary molybdenum    on rat and calf liver and heart enzymes,” J. Nutr., 70:63, 1960.-   Culotta, Klomp, Strain, Casareno, Krems and Gitlin, “The copper    chaperone for superoxide dismutase,” J. Biol. Chem. 272:23469-23472,    1997.-   Danks, “Disorders of Copper Transport,” In: Metabolic Basis of    Inherited Diseases, Vol. I, Sixth Ed., Scriver, Beaudet, Sly, Valle    (Eds.), McGraw Hill, New York, pp. 1411-1431, 1989.-   Dick and Bull, “Some preliminary observations of the effect of    molybdenum on copper metabolism in herbivorous animals,” Aust. Vet.    J., 21:70, 1945.-   Dick, Dewey, Gawthome, “Thiomolybdates and the    copper-molybdenum-sulphur interaction in ruminant nutrition,” J.    Agri. Sci., 85:567, 1975.-   Dvorak et al., “Distribution of vascular permeability factor    (vascular endothelial growth factor) in tumors: concentration in    tumor blood vessels,” J. Exp. Med., 174:1275-1278, 1991.-   Engleka and Maciag, “Inactivation of human fibroblast growth    factor-1 (FGF-1) activity by interaction with copper ions involves    FGF-1 dimer formation induced by copper-catalyzed oxidation,” J.    Biol. Chem. 267:11307-11315, 1994.-   Epenetos et al., “Limitations of radiolabeled monoclonal antibodies    for localization of human neoplasms,” Cancer Res., 46:3183-3191,    1986.-   Fell, Dinsdale, El-Gallad, “Gut Pathology of Rats Dosed with    Tetrathiomolybdate,” J. Com. Pathol. 89:495, 1979.-   Ferguson, Lewis, Waterson, “The Teart Pastures of Somerset I. The    Cause and Cure of Teartness,” J. Agr. Sci. 33:44, 1943.-   Folkman, “Angiogenesis in cancer, vascular, rheumatoid and other    disease,” Nature Med. 1:27-31, 1995c.-   Folkman, “Angiogenesis inhibitors generated by tumors,” Mol. Med.,    1(2):120-122, 1995a.-   Folkman, “Anti-angiogenesis: new concept for therapy of solid    tumors,” Ann. Surg. 175:409-416, 1972.-   Folkman, “The influence of angiogenesis research on management of    patients with breast cancer,” Breast Cancer Res. Treat.,    36(2):109-118, 1995b.-   Folkman, In: Cancer: Principles and Practice of Oncology,    Lippincott-Raven Publishers, pp. 3075-3085, 1997.-   Fuchs and Sacerdote de Lustig, “Localization of tissue copper in    mouse mammary tumors,” Oncol. 46:183-187, 1989.-   Glass, Reich, DeLong, “Wilson's Disease: Development of Neurological    Disease After Beginning Penicillamine Therapy,” Arch. Neurol.    47:595-596, 1990.-   Gooneratne, Howell, Gawthorne, “An investigation of the effects of    innvenous administration of thiomolybdate on copper metabolism in    chronic Cu-poisoned sheep,” Br. J. Nutr., 46:469, 1981b.-   Gooneratne, Howell, Gawthome, “Intravenous Administration of    Thiomolybdate for the Prevention and Treatment of Chronic Copper    Poisoning in Sheep,” Br. J. Nutr. 46:457, 1981a.-   Gullino, “Considerations on the mechanism of the angiogenic    response,” Anticancer Res., 6(2):153-158, 1986.-   Gullino, Ziche and Alessandri, “Gangliosides, Copper ions and    angiogenic capacity of adult tissues,” Cancer Metastasis Rev.    9:239-251, 1990.-   Guo, Krutzch, Inman and Roberts, “Thrombospondin 1 and type I repeat    peptides of thrombospondin 1 specifically induce apoptosis of    endothelial cells,” Cancer Res. 57:1735-1742, 1997.-   Guy, Webster, Schaller, Parsons, Cardiff, Muller, “Expression of the    neu protooncogene in the mammary epithelium of transgenic mice    induces metastatic disease,” Proc. Natl. Acad. Sci. USA,    89:10578-10582, 1992.-   Hadjikyriacou and Coucouvanis, “New members of the    [MO₂(S)_(n)(S₂)_(6-n)]²⁻ series. Synthesis, structural    characterization, and properties of the [MO₂S₉]²⁻, [Mo₂S₇]²⁻ and    [Mo₂S₆]²⁻ thioanions,” Inorg. Chem. 26:2400-2408, 1987.-   Hanahan and Folkman, “Patterns and emerging mechanisms of the    angiogenic switch during tumorigenesis,” Cell, 86(3):353-364, 1996.-   Harper and Walshe, “Reversible Pancytopenia Secondary to Treatment    with Tetrathiomolybdate,” Br. J. Haematol. 64:851-853, 1986.-   Hayes, “Angiogenesis and breast cancer,” Hematol. Oncol. Clin. North    Am., 8(1):51-71, 1994.-   Hill, Brewer, Juni, Prasad, Dick, “Treatment of Wilson's Disease    with Zinc II. Validation of Oral ⁶⁴Copper Uptake with Copper    Balance,” Am. J. Med. Sci. 12:344, 1986.-   Hill, Brewer, Prasad, Hydrick, Hartmann, “Treatment of Wilson's    disease with zinc, I: oral zinc therapy regimens,” Hepatology,    7:522-528, 1987.-   Hoogenraad, Koevoet, De Ruyter Korver, “Oral zinc sulfate as    long-term treatment in Wilson's disease (hepatolenticular    degeneration),” Eur. Neurol. 18:205-211, 1979.-   Hoogenraad, Van den Hamer, Koevoet, De Ruyter Korver, “Oral zinc in    Wilson's disease,” Lancet 2:1262-1263, 1978.-   Hoogenraad, Van Hattum, Van den Hamer, “Management of Wilson's    disease with zinc sulfate: Experience in a series of 27    patients,” J. Neurol. Sci. 77:137-146, 1987.-   Horak, Harris, Stuart, Bicknell, “Angiogenesis in breast cancer.    Regulation, prognostic aspects, and implications for novel treatment    strategies,” Ann. NY Acad. Sci., 698:71-84, 1993.-   Humphries, Mills, Greig, Roberts, Inglis, Halliday, “Use of Ammonium    Tetrathiomolybdate in the Treatment of Copper Poisoning in Sheep,”    Vet. Record 119:596-598, 1986.-   Humphries, Morrice, Bremner, “A Convenient Method for the Treatment    of Chronic Copper Poisoning in Sheep using Subcutaneous Ammonium    Tetrathiomolybdate,” Vet. Record 123:51-53, 1988.-   Hynes, Lamand, Montel, Mason, “Some Studies on the Metabolism and    the Effects of ⁹⁹Mo- and ³⁵S-Labelled Thiomolybdates After    Intravenous Infusion in Sheep,” Br. J. Nutr. 52:149, 1984.-   Ingber et al., “Angioinhibins: Synthetic analogues of fumagillin    which inhibit angiogenesis and suppress tumor growth,” Nature,    48:555-557, 1990.-   Iruela-Arispe and Dvorak, “Angiogenesis: A dynamic balance of    stimulators and inhibitors,” Thromb. Haemost. 78:672-677, 1997.-   Jacob, Sanstead, Munoz, Klevay, Milne, “Whole body surface loss of    trace metals in normal males,” Am. J. Clin. Nutr., 34:1379-1383,    1981.-   Jain, “Vascular and interstitial barriers to delivery of therapeutic    agents in tumors,” Cancer Metastasis Rev., 9:253-266, 1990.-   Jones, Gooneratne, Howell, “X-ray Microanalysis of Liver and Kidney    in Copper Loaded Sheep with and without Thiomolybdate    Administration,” Res. Vet. Sci. 37-273, 1984.-   Juweid et al., “Micropharmacology of monoclonal antibodies in solid    tumors: direct experimental evidence for a binding site barrier,”    Cancer Res., 52:5144-5153, 1992.-   Kanatzidis and Coucouvanis, “Structure of Bis(tetraethylammonium)    tetrathiomolybdate(VI), 2C₈H₂₀N⁺MoS₄ ²⁻ ,” Acta Cryst. C39:835-838,    1983.1983.-   Lannutti, Gately, Quevedo, Soff and Paller, “Human angiostatin    inhibits murine hemangioendothelioma tumor growth in vivo,” Cancer    Res. 57:5277-5280, 1997.-   LeCarpentier, Tridandapani, Fowlkes, Roubidoux, Moskalik and Carson,    “Utility of 3D ultrasound in the discrimination and detection of    breast cancer,” Rad. Soc, North Amer. EJ, 1999.-   Lee, Brewer, Wang, “The Treatment of Wilson's Disease with Zinc VII.    Protection of the Liver for Copper Toxicity by Zinc Induced    Metallothionein in a Rat Model,” J. Lab. Clin. Med. 114:639-645,    1989.-   Linder, Houle, Isaacs, Moor and Scott, “Copper regulation of    ceruloplasmin in copper-deficient rats,” Enzyme 24:23-35, 1979.-   Lowder et al., “Studies on B lymphoid tumors treated with monoclonal    anti-idiotype antibodies: correlation with clinical responses,”    Blood, 69:199-210, 1987.-   Macilese Ammerman, Valsecchi, Dunavant, Davis, “Effect of dietary    molybdenum and sulfate upon copper metabolism in sheep,” J. Nutr.,    99:177, 1969.-   Marshall, Wellstein, Rae, DeLap, Phipps, Hanfelt, Yunmbam, Sun,    Duchin and Hawkins, “Phase I trial of orally administered pentosan    polysulfate in patients with advanced cancer,” Clin. Cancer Res.    3:2347-2354, 1997.-   Mason, “The biochemical pathogenesis of molybdenum-induced copper    deficiency syndromes in ruminants: Towards the final chapter,” Irish    Veter. J., 43:18-21, 1990.-   Mason, Lamand, Hynes, “⁹⁹Mo Metabolism in Sheep After the    Intravenous Injection of ⁹⁹Mo Thiomolybdates,” J. Inorg. Biochem.    19:153, 1983.-   McQuaid and Mason, “A comparison of the effects of penicillamine,    trientine, and trithiomolybdate on [³⁵S]-labeled metallothionein in    vitro; the implications for Wilson's disease therapy,” J. Inorg.    Biochem., 41:87-92, 1991.-   Merajver, Irani, van Golen and Brewer, “Copper depletion as an    anti-angiogenic strategy in HER2-neu transgenic mice,” Proceedings    of Special AACR Conference on Angiogenesis and Cancer, Abstract    #B-11, Jan. 22-24, 1998.-   Millauer, Longhi, Plate, Shawver, Risau, Ullrich and Strawn,    “Dominant-negative inhibition of Flk-1 suppresses the growth of many    tumor types in vivo,” Cancer Res. 56:1615-1620, 1996.-   Miller and Engel, “Interrelations of copper, molybdenum; and sulfate    sulfur in nutrition,” Fed. Proc., 19:666, 1960.-   Mills, El-Gallad, Bremner, “Effects of molybdate, sulfide, and    tetrathiomolybdate on copper metabolism in rats,” J. Inorg.    Biochem., 14:189, 1981a.-   Mills, El-Gallad, Bremner, Wenham, “Copper and molybdenum absorption    by rats given ammonium tetrathiomolybdate,” J. Inorg. Biochem., 14:    163, 1981b.-   Mills, Monty, Ichihara, Pearson, “Metabolic effects of molybdenum    toxicity in the rat,” J. Nutr., 65:129, 1958.-   Muller and Krickemeyer, Inorg. Synth. 27:47, 1990.-   Muller, Nolte and Krebs, Inorg. Chem. 19:2835, 1980.-   Muller, Sinn, Pattengale, Wallace, Leder, “Single-step induction of    mammary adenocarcinoma in transgenic mice bearing the activated    c-neu oncogene,” Cell, 54:105-115, 1988.-   O'Reilly, Boehm, Shing, Fukai, Vasios, Lane, Flynn, Birkhead, Olsen    and Folkman, “Endostatin: An endogenous inhibitor of angiogenesis    and tumor growth,” Cell 88:277-285, 1997.-   O'Reilly, Holmgren, Shing, Chen, Rosenthal, Moses, Lane, Cao, Sage    and Folkman, “Angiostatin: A novel angiogenesis inhibitor that    mediates the suppression of metastases by a Lewis lung carcinoma,”    Cell 79:315-328, 1994.-   Parangi, O'Reilly, Christofori, Holmgren, Grosfeld, Folkman,    Hanahan, “Antiangiogenic therapy of transgenic mice impairs de novo    tumor growth,” Proc. Natl. Acad. Sci. USA, 93(5):2002-2007, 1996.-   Parke, Bhattacherjee, Palmer, Lazarus, “Characterization and    quantification of copper sulfate-induced vascularization of the    robbit cornea,” Am. J. Pathol., 137:173-178, 1988.-   Patstone and Maher, “Copper and calcium binding motifs in the    extracellular domains of fibroblast growth factor receptors,” J.    Biol. Chem. 271:3343-3346, 1996.-   Qian, Wang, Rothman, Nicosia and Tuszynski, “Thrombospondin-1    modulates angiogenesis in vitro by up-regulation of matrix    metalloproteinase-9 endothelial cells,” Exp. Cell Res. 235:403-412,    1997.-   Raju, Alessandri, Ziche, Gullino, “Ceruloplasmin, Copper Ions, and    Angiogenesis,” J. Natl. Cancer Inst. 69:1183-1188, 1982.-   Remington's Pharmaceutical Sciences, 15th Ed., Mack Publishing    Company, 1975.-   Remington's Pharmaceutical Sciences, 16th Ed., Mack Publishing    Company, 1980.-   Salnikow, Wang and Costa, “Induction of activating transcription    factor 1 by Nickel and its role as a negative regulator of    thrombospondin I gene expression,” Cancer Res. 57:5060-5066, 1997.-   Sands, Immunoconjugates and Radiopharmaceuticals, 1:213-226, 1988.-   Schapira and Schapira, “Use of ceruloplasmin levels to monitor    response to therapy and predict recurrence of breast cancer,” Breast    Cancer Res Treat. 3:223-224, 1983.-   Scheinberg and Sternlieb, “Wilson's Disease,” In: Major Problems in    Internal Medicine, Vol. XXIII, W.B. Saunders Company, Philadelphia,    1984.-   Seelig, “Review: Relationships of Copper and Molybdenum to Iron    Metabolism,” Am. J. Clin. Nutr. 25:1022, 1972.-   Shing, “Heparin-copper biaffinity chromatography of fibroblast    growth factors,” J. Biol. Chem. 263:9059-9062, 1988.-   Shockley et al., “Penetration of tumor tissue by antibodies and    other immunoproteins,” Ann. N.Y. Acad. Sci., 617:367-382, 1991.-   Sim, O'Reilly, Liang, Fortier, He, Madsen, Lapcevich and Nacy, “A    recombinant human angiostatin protein inhibits experimental primary    and metastatic cancer,” Cancer Res. 57:1329-1334, 1997.-   Suzuki, Yamamoto, Aoki, Takeichi, “Selective removal of copper bound    to metalliothionein in the liver of LEC rats by tetrathiomolybdate,”    TOXIC 83:149, 1993.-   Takahashi, Mulliken, Kozakewich, Rogers, Folkman and Ezekowitz,    “Cellular markers that distinguish the phases of hemangioma during    infancy and childhood,” J. Clin. Invest. 93:2357-2364, 1994.-   Teo et al., “Mo, W, and Fe EXAFS of the [Cl₂FeS₂MS₂FeCl₂]²⁻    (M=Mo, W) dianions. A comparison with the Mo EXAFS of    nitrogenase,” J. Am. Chem. Soc. 105:5767-5770, 1983.-   Vitetta et al., “Phase I immunotoxin trial in patients with B-cell    lymphoma,” Cancer Res., 15:4052-4058, 1991.-   Volpert, Stellmach and Bouck, “The modulation of thrombospondin and    other naturally occurring inhibitors of angiogenesis during tumor    progression,” Breast Cancer Res. Treat., 36:119-126, 1995.-   Volpert, Ward, Lingen, Chesler, Solt, Johnson, Molteni, Polverini    and Bouck, “Captopril inhibits angiogenesis and slows the growth of    experimental tumors in rats,” J. Clin. Invest. 98:671-679, 1996.-   Walshe, “Penicillamine: A New Oral Therapy for Wilson's Disease,”    Am. J. Med. 21:487, 1956.-   Walshe, “Treatment of Wilson's disease with trientine (triethylene    tetramine) dihydrochloride,” Lancet, 1:643-647, 1982.-   Warren, Yuan, Malti, Gillett and Ferrara, “Regulation by vascular    endothelial growth factor of human colon cancer tumorigenesis in a    mouse model of experimental liver metastasis,” J. Clin. Invest.    95:1789-1797, 1995.-   Watanabe, Seno, Sasada and Igarashi, “Molecular characterization of    recombinant human acidic fibroblast growth factor produced in E.    coli: Comparative studies with human basic fibroblast growth    factor,” Mol. Endo. 4:869-879, 1990.-   Wu, Forbes, Chen, Cox, “The LEC rat has a deletion in the copper    transporting ATPase gene homologous to the Wilson disease gene,”    Nat. Genet. 7:541, 1994.-   Yoshida, Ikeda, Nakazawa, “Copper chelation inhibits tumor    angiogenesis in the experimental 9L gliosarcoma model,”    Neurosurgery, 37(2):287-292, 1995.-   Young, Shoulson, Penney et al., “Huntington's Disease in Venezuela:    Neurologic Features and Functional Decline,” Neurol. 36:244-249,    1986.-   Yuan, Chen, Dellian, Safabakhsh, Ferrara and Jain, “Time-dependent    vascular regression and permeability changes in established human    tumor xenografts induced by an anti-vascular endothelial growth    factor/vascular permeability factor antibody,” Proc. Natl. Acad.    Sci. USA 93:14765-14770, 1996.-   Yuzbasiyan-Gurkan, Brewer, Abrams, Main, Giacherio, “Treatment of    Wilson's Disease with Zinc V. Changes in Serum Levels of Lipase,    Amylase and Alkaline Phosphatase in Wilson's Disease Patients,” J.    Lab. Clin. Med. 114:520-526, 1989.-   Yuzbasiyan-Gurkan, Grider, Nostrant, Cousins, Brewer, “The Treatment    of Wilson's Disease with Zinc X. Intestinal Metallothionein    Induction,” J. Lab. Clin. Med. 120:380-386, 1992.-   Ziche, Jones, Gullino, “Role of Prostaglandin E₁ and Copper in    Angiogenesis,” J. Natl. Cancer Inst. 69:475-482, 1982.

1. A method of treating a disease characterized by ocularneovascularization in an animal, comprising orally administering to ananimal having a disease characterized by ocular neovascularization aloading dose of greater than 200 mg daily up to 410 mg daily of athiomolybdate compound that binds copper and forms thiomolybdatecompound-copper-protein complex.
 2. The method of claim 1, wherein saidthiomolybdate compound comprises at least a first iron atom.
 3. Themethod of claim 1, wherein said thiomolybdate compound comprises atleast a first oxygen atom.
 4. The method of claim 1, wherein saidthiomolybdate compound is associated with at least a first carbohydratemolecule.
 5. The method of claim 4, wherein said carbohydrate moleculeis a disaccharide molecule.
 6. The method of claim 4, wherein saidcarbohydrate molecule is a sucrose molecule.
 7. The method of claim 6,wherein said thiomolybdate compound is associated with about 30 sucrosemolecules.
 8. The method of claim 1, wherein said thiomolybdate compoundis dodecathiodimolybdate, tetrathiomolybdate, iron octathiodimolybdate,trithiomolybdate, dithiomolybdate or monothiomolybdate.
 9. The method ofclaim 8, wherein said thiomolybdate compound is dodecathiodimolybdate.10. The method of claim 8, wherein said thiomolybdate compound is ironoctathiodimolybdate.
 11. The method of claim 8, wherein saidthiomolybdate compound is tetrathiomolybdate.
 12. The method of claim 1,further comprising administering to said animal a therapeuticallyeffective amount of a zinc compound.
 13. The method of any one of claims2 through 11, wherein said animal is a human subject.
 14. The method ofclaim 13, wherein said thiomolybdate compound is administered to saidhuman subject in an amount and for a time effective to reduce the levelof copper in said human subject to between about 40% and about 10% ofthe level of copper in said human subject prior to administration ofsaid thiomolybdate compound.
 15. The method of claim 14, wherein saidthiomolybdate compound is administered to said human subject in anamount and for a time effective to reduce the level of copper in saidhuman subject to about 20% of the level of copper in said human subjectprior to administration of said thiomolybdate compound.
 16. The methodof claim 14, comprising: a) administering said thiomolybdate compound tosaid human subject in an amount and for a time effective to reduce thelevel of copper in said human subject to about 20% of the level ofcopper in said human subject prior to administration of saidthiomolybdate compound; and b) administering to said human subject atherapeutically effective amount of a zinc compound.
 17. The method ofclaim 16, wherein said therapeutically effective amount of a zinccompound is administered to said human subject for a period of timeeffective to maintain the level of copper in said human subject at about20% of the level of copper in said human subject prior to administrationof said thiomolybdate compound.
 18. The method of claim 14, wherein thelevel of copper in said human subject is indicated by the level of serumceruloplasmin.
 19. The method of claim 1, wherein said disease isassociated with corneal neovascularization.
 20. The method of claim 19,wherein said disease is epidemic keratoconjunctivitis, Vitamin Adeficiency, contact lens overwear, atopic keratitis, superior limbickeratitis, pterygium keratitis sicca, sjogrens, acne rosacea,phylectenulosis, syphilis, Mycobacteria infections, lipid degeneration,chemical bums, bacterial ulcers, fungal ulcers, Herpes simplexinfections, Herpes zoster infections, protozoan infections, Kaposisarcoma, Mooren ulcer, Terrien's marginal degeneration, marginalkeratolysis, trauma, Scleritis, Steven's Johnson disease, periphigoidradial keratotomy or corneal graft rejection.
 21. The method of claim 1,wherein said disease is associated with retinal/choroidalneovascularization.
 22. The method of claim 21, wherein said disease isdiabetic retinopathy, sickle cell anemia, pseudoxanthoma elasticum,Pagets disease, vein occlusion, artery occlusion, carotid obstructivedisease, chronic uveitis/vitritis, Lyme's disease, pars planitis,chronic retinal detachment, hyperviscosity syndromes, toxoplasmosis,trauma or post-laser complication.
 23. The method of claim 21, whereinsaid disease is associated with choroidal neovascularization.
 24. Themethod of claim 23, wherein said disease is age-related maculardegeneration, dry type macular degeneration, ocular histoplasmosissyndrome, pathologic myopia or angioid streaks.
 25. The method of anyone of claims 19 through 24, wherein said animal is a human subject.